EP2235194A2 - Method for fermentatively producing 1,5-diaminopentane - Google Patents

Method for fermentatively producing 1,5-diaminopentane

Info

Publication number
EP2235194A2
EP2235194A2 EP09704118A EP09704118A EP2235194A2 EP 2235194 A2 EP2235194 A2 EP 2235194A2 EP 09704118 A EP09704118 A EP 09704118A EP 09704118 A EP09704118 A EP 09704118A EP 2235194 A2 EP2235194 A2 EP 2235194A2
Authority
EP
European Patent Office
Prior art keywords
dap
lysine
fermentation broth
fermentation
comonomer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP09704118A
Other languages
German (de)
French (fr)
Other versions
EP2235194B1 (en
Inventor
Martin VÖLKERT
Oskar Zelder
Burkhard Ernst
Weol Kyu Jeong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Priority to PL09704118T priority Critical patent/PL2235194T3/en
Priority to EP09704118A priority patent/EP2235194B1/en
Publication of EP2235194A2 publication Critical patent/EP2235194A2/en
Application granted granted Critical
Publication of EP2235194B1 publication Critical patent/EP2235194B1/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
    • C07C209/82—Purification; Separation; Stabilisation; Use of additives
    • C07C209/84—Purification

Definitions

  • the present invention relates to a process for the isolation of 1, 5-diaminopentane (DAP) from DAP-containing fermentation broths, a process for the fermentative production of DAP using this isolation method and a process for the preparation of DAP-containing polymers using the in this way isolated or fermentatively produced DAPs.
  • DAP 1, 5-diaminopentane
  • 1,5-Diaminopentane (often also referred to as pentamethylenediamine or cadaverine, hereinafter referred to as DAP) represents an important raw material of the chemical industry.
  • DAP is used in the production of polyamides, polyureas or polyurethanes and copolymers thereof.
  • EP-A-1 482 055 describes the enzymatic decarboxylation of lysine in the presence of a dicarboxylic acid for adjusting the pH during the reaction.
  • the DAP dicarboxylate obtained in the preparation is isolated by first destaining the concentrated solution with activated charcoal, concentrating and crystallizing out DAP dicarboxylate by cooling crystallization.
  • WO-A-2006/123778 describes the preparation of DAP carbonate by enzymatic decarboxylation of lysine in the presence of carbon dioxide. Concentration of the reaction solution and removal of carbon dioxide DAP is formed.
  • JP 2004-208 646 describes the preparation of DAP dicarboxylate by enzymatic decarboxylation of a solution containing L-lysine dicarboxylate and precipitation of DAP dicarboxylate by addition of an organic solvent selected from among alcohols, ketones and nitriles.
  • JP 2004-222 569 describes the production of DAP using an L-lysine decarboxylase-expressing coryneform bacterium, setting the KuI supernatant to pH 12 and extraction of DAP with a polar organic solvent.
  • JP 2004-000114 describes the preparation of DAP by reacting highly concentrated L-lysine monohydrochloride with L-lysine decarboxylase-expressing E. coli cells, adjusting the reaction solution to pH ⁇ 13 and extracting the reaction product with one polar organic solvent and subsequent distillation.
  • the present invention is therefore based on the object of further improving the isolation of DAP (cadaverine) from fermentation broths.
  • DAP cadaverine
  • the yield of valuable material should be further increased and the time required for the isolation, in particular the solvent-based extraction, improved.
  • this object has been achieved by providing a process in which the treated to an alkaline pH fermentation broth is thermally treated and then extracted with a suitable organic extractant. It was surprisingly found that DAP-containing by-products of the fermentation, especially acetyl-DAP are hydrolytically cleaved to release the desired product and, surprisingly, the rate of phase separation during the extraction step can be significantly increased. The increased rate of phase separation is particularly evident in the processing of fermentation broths from the fermentation of microorganisms in the presence of complex nutrient media, such as yeast extract. DESCRIPTION OF THE FIGURES
  • FIG. 1 shows a block diagram for the course of a specific embodiment according to the invention of an overall process for isolating DAP from a fermentation broth.
  • Figure 2 illustrates the hydrolytic cleavage of acetyl-DAP (triangles) to form DAP (diamonds) over a five hour thermal treatment of a fermentation broth by refluxing at pH 13.7. The content of residual lysine (squares) remains unchanged during the thermal treatment.
  • Figure 3 illustrates the release of ammonia during the heating process and the subsequent refluxing of the fermentation broth.
  • the invention relates to a method for the isolation of 1, 5-diaminopentane (DAP) from a DAP-containing fermentation broth, wherein the fermentation broth a) alkalized, b) thermally treated, c) extracted DAP with an organic extractant, and d) DAP isolated from the separated organic phase.
  • DAP 1, 5-diaminopentane
  • the fermentation broth to a pH of> 1 1, in particular ⁇ 1 1, 5 or ⁇ 12, in particular ⁇ 12 to 14, or 12.5 to 13.8, or 13 to 13.8, or 13.5 to 13.7.
  • the pH adjustment is carried out in particular by adding an alkali metal or alkaline earth metal hydroxide, such as a Na, K or Ca hydroxide.
  • the material distribution can be further optimized by adjusting the pH, with optimum mass transfer conditions being able to be set at pH values above about 12.5.
  • cleavage of optionally contained acetyl-DAP can be further optimized by adjusting the pH, wherein - depending on the amount of contained acetyl-DAP - can be adjusted at pH values above about 13 optimal cleavage conditions (cleavage kinetics).
  • cellular components may be removed from the fermentation broth prior to alkalinization.
  • Methods for removing the cellular components are well known to those skilled in the art (e.g., separators, decanters, flocculation, filtration methods, or combinations of several such process steps).
  • the alkalized fermentation broth is thermally treated by passing, for example batchwise or continuously, to reflux temperature, such as 90-1 10 0 C at atmospheric pressure or to a higher temperature at overpressure, such as 0- 100 bar, in particular 0-25 bar overpressure, heated.
  • the thermal treatment is carried out under conditions which cause a, preferably substantially quantitative, hydrolytic cleavage of optionally present acetyl-DAP.
  • the person skilled in the art can coordinate the essential process parameters, such as pressure, temperature and residence time, as required.
  • acetyl-DAP includes mono- and di-acetyl-DAP, but usually the mono-acetyl form is predominantly present In a further embodiment, this heating can be carried out in several stages, eg also with recovery of the liberated ammonia by intermediate relaxation.
  • the DAP extraction is carried out with an organic solvent with miscibility gap with water which is as polar as possible and stable in the alkaline, in particular a polar, in particular dipolar protic, organic solvent.
  • organic solvent with miscibility gap with water which is as polar as possible and stable in the alkaline, in particular a polar, in particular dipolar protic, organic solvent.
  • Suitable solvents are described in a following section
  • the DAP extraction and / or the subsequent phase separation is carried out batchwise at elevated temperature. Further embodiments of the extraction and the workup of the DAP-containing extract are described in a following section.
  • the process according to the invention is suitable for the processing of fermentation broths from the fermentation of a microorganism in a complex culture medium, such as, for example, yeast extract-containing culture medium.
  • a complex culture medium such as, for example, yeast extract-containing culture medium.
  • Another object of the invention is a process for the fermentative production of DAP, wherein a lysine-producing microorganism is cultured under lysine and optionally DAP-producing conditions and the resulting DAP isolated using a DAP isolation method as defined above.
  • the fermentation can be carried out in a culture medium with complex media components.
  • a lysine-producing microorganism which additionally lysine decarboxylase activity, such as. a heterologous, i. from another organism, lysine decarboxylase (LDC) expressed.
  • LDC lysine decarboxylase
  • Complex nutrient or culture media or “complex media” are, according to the invention, media known per se which comprise mixtures of substances of complex composition, such as e.g. Corn steep liquor, tryptone, bactone, soy hydrolyzate and, in particular, yeast extract.
  • a lysine-containing fermentation broth may be contacted with purified, optionally immobilized lysine decarboxylase to decarboxylate lysine to DAP, or another optionally immobilized LDC-expressing microorganism may be added to or contacted with the broth.
  • Suitable processes are described in the prior art, to which reference is hereby expressly made. (See, for example, JP 2002-223771).
  • the entire isolation process described above or the fermentative preparation process described above or individual steps thereof can be carried out continuously or discontinuously, in batch or semibatch, or fed-batch or repeated (fed) batch mode.
  • Another object of the invention relates to a process for the preparation of a DAP-containing polymer, wherein first prepared monomeric DAP by a process according to the above definition by fermentation and isolated and polymerized together with at least one other comonomer.
  • the comonomer may in particular be selected from polycarboxylic acids, in particular dicarboxylic acids with 4 to
  • Nonlimiting examples of suitable dicarboxylic acids are Bern nonlimiting examples of suitable diisocyanates are methylene diphenyl diisocyanate (MDI), toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI) and isophorone diisocyanate.
  • MDI methylene diphenyl diisocyanate
  • TDI toluene diisocyanate
  • HDI hexamethylene diisocyanate
  • isophorone diisocyanate are particularly, polymers of the polyamide, polyurea or polyurethane type are formed, for example polyamide 5, 10 or polyamide 5, 6.
  • At least one comonomer is added to the isolated DAP or a mixture of DAP and at least one comonomer from a DAP precipitation is used.
  • a suitable DAP / comonomer mixture from a salt precipitation of DAP described above may result from a distilled reclaimed DAP extract.
  • the comonomer is preferably a polycarboxylic acid, such as sebacic acid.
  • the present invention is basically applicable to the processing of any DAP-containing fermentation broths.
  • the microorganisms used in the fermentation may be naturally occurring, improved by mutation and selection, but especially recombinant microorganisms such as fungi, but especially bacteria.
  • These microorganisms have either the direct ability to produce DAP and / or DAP derivatives, such as acetyl-DAP, but at least they are capable of fermentative production of lysine, especially L-lysine.
  • DAP pathway diaminopimelate pathway
  • succinylase pathway or the dehydrogenase pathway is capable.
  • microorganisms can produce lysine, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol, fatty acids or vegetable oils or ethanol and preferably at least partially release the lysine formed into the extracellular space.
  • these are coryneform bacteria, in particular the genus Corynebacterium or the genus Brevibacterium. From the genus Corynebacterium in particular the species Corynebac- terium glutamicum, which is known in the art for its ability to produce L-amino acids.
  • suitable strains of coryneform bacteria are those of the genus Corynebacterium, in particular of the species Corynebacte ⁇ um glutamicum (C. glutamicum), such as
  • Brevibacterium flavum ATCC 14067 Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC 14020;
  • the abbreviation KFCC means the Korean Federation of Culture Collection
  • the abbreviation ATCC denotes the American strain strain culture collection
  • abbreviated FERM BP the collection of the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan ,
  • Fermentation broths to be worked up according to the invention originate, for example, from the cultivation of recombinant coryneform bacteria, which produce a lysin biosynthesis-promoting deregulatory intervention involving at least one lysine biosynthesis gene, lysine, in particular L-lysine or a lysine-containing substance mixture and / or which additionally an enzyme with lysine decarbo- overexpressing xylase activity and accumulating DAP and / or acetyl-DAP.
  • the latter are thus capable of direct DAP production.
  • the gene involved in lysine biosynthesis is known from WO 2007/113127, to which reference is hereby expressly made.
  • “deregulation” is to be understood in the broadest sense and encompasses both increasing or decreasing or shutting down enzyme activity in a variety of ways, eg by increasing or decreasing the copy number of enzyme molecules in the microorganism or changing another one which reduces lysine biosynthesis Property.
  • the enzyme lysine decarboxylase (E.C. 4.1.1.18.) Catalyzes the decarboxylation of L-lysine to DAP.
  • the enzyme is, for example, the cadA gene product (Kyoto Encyclopedia of Genes and Genomes, Entry b4131) or the IdcC gene product (Kyoto Encyclopedia of Genes and Genomes, Entry JW0181).
  • cadA gene product Kyoto Encyclopedia of Genes and Genomes, Entry b4131
  • IdcC gene product Korean of Genes and Genomes, Entry JW0181.
  • Their use for the production of recombinant microorganisms for cadaverine production are known to the person skilled in the art (cf., for example, EP-A-1 482 055).
  • the skilled person can take different measures individually or in combination.
  • the copy number of the corresponding genes can be increased, or the promoter and regulatory region or ribosome binding site located upstream of the structural gene can be mutated.
  • expression cassettes which act upstream be incorporated into the structural gene.
  • inducible promoters it is additionally possible to increase expression in the course of fermentative L-lysine production. Measures to extend the lifetime of mRNA also improve expression.
  • enzyme activity is also enhanced.
  • the genes or gene constructs can either be present in one or more plasmids with different copy numbers or be integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can be achieved by changing the composition of the medium and culture.
  • expression constructs or vectors which contain, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a desired enzyme activity.
  • such constructs comprise a promoter 5'-upstream of the respective coding sequence and a terminator sequence 3'-downstream, and optionally other common regulatory elements, each operably linked to the coding sequence.
  • "Operational linkage” is understood to mean the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended Enhancers and the like Further regulatory elements include selectable markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in Goeddel, GE. Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the natural regulatory sequence may still be present before the actual structural gene. By genetic modification, this natural regulation can optionally be switched off and the expression of the genes increased or decreased.
  • the gene construct can also be constructed more simply, that is, no additional regulatory signals are inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation stops and gene expression is increased or decreased.
  • the nucleic acid sequences may be contained in one or more copies in the gene construct.
  • promoters examples include: the promoters, ddh, amy, lysC, dapA, lysA from Corynebacterium glutamicum, but also gram-positive promoters SPO2 as described in Bacillus Subtilis and Its Closest Relatives, Sonenshein, Abraham L., Hoch, James A. , Losick, Richard; ASM Press, District of Columbia, Washington and Patek M. Eikmanns BJ. Patek J. Sahm H. Microbiology.
  • inducible promoters such as light and in particular temperature-inducible promoters, such as the P r P r promoter.
  • synthetic promoters such as multiple promoters (cf., for example, WO2006 / 069711) can also be used to advantage.
  • the said regulatory sequences are intended to enable targeted expression of the nucleic acid sequences. Depending on the host organism, this may mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can thereby preferably positively influence the expression and thereby increase or decrease.
  • an enhancement of the regulatory elements can advantageously take place at the level of transcription, by using strong transcription signals such as promoters and / or enhancers. be used.
  • an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
  • An expression cassette is produced by fusion of a suitable promoter, a suitable Shine-Dalgarnow sequence with a lysine biosynthesis nucleotide sequence and a suitable termination signal.
  • a suitable promoter a suitable Shine-Dalgarnow sequence
  • a suitable termination signal a suitable termination signal.
  • Common recombinant and cloning techniques are used, as described, for example, in Current Protocols in Molecular Biology, 1993, John Wiley & Sons, Incorporated, New York New York, PCR Methods, Gelfand, David H., Innis, Michael A., Sninsky, John J. 1999, Antibacterial Press, Incorporated, California, San Diego, PCR Cloning Protocols, Methods in Molecular Biology Ser., Vol. 192, 2nd ed., Humana Press, New Jersey, Totowa. T.
  • the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector for expression in a suitable host organism, which enables optimal expression of the genes in the host.
  • Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985).
  • vectors include all other vectors known to the person skilled in the art, such as, for example, phages, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally.
  • Suitable plasmids are those which are replicated in coryneform bacteria.
  • Numerous known plasmid vectors such as PZKE (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExi (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al , Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
  • Other plasmid vectors such as. B.
  • pCLiK5MCS or those based on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891), can be in be used in the same way.
  • those plasmid vectors by means of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB operon.
  • the complete gene is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum.
  • plasmid vectors which are used are pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), Bernard et al.
  • the plasmid vector containing the gene to be amplified is then transformed into the desired strain of C. glutamicum by transformation. Methods for transformation are described by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Biotechnology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123,343-347 (1994)).
  • Enzymes can be affected in their activity by mutations in the corresponding genes such that there is a partial or complete reduction in the reaction rate of the enzymatic reaction.
  • mutations are known to those skilled in the art (Motoyama H. Yano H. Terasaki Y. Anazawa H. Applied & Environmental Microbiology 67: 3064-70, 2001, Eikmanns BJ, Eggeling L. Sahm H. Antonie van Leeuwenhoek 64: 145 -63, 1993-94.). With this measure, e.g. With the lysine biosynthesis according to the invention competing reactions are eliminated or slowed down. (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
  • L-lysine it may be advantageous, in addition to expression or amplification of the lysine biosynthesis genes, to have one or more enzymes of an upstream biosynthetic pathway, such as e.g. pentose-phosphate metabolism, the citric acid cycle, or amino acid export.
  • an upstream biosynthetic pathway such as e.g. pentose-phosphate metabolism, the citric acid cycle, or amino acid export.
  • the microorganisms used according to the invention can be used continuously or discontinuously in the batch process (batch cultivation) or in the fed batch (feed process). Ren) or repeated fed batch process (repetitive feed method) for the production of L-lysine, be cultured.
  • a summary of known cultivation methods is in the textbook by Chmiel (Bioreatechnik 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) Find.
  • the culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society of Bacteriology (Washington D.C, USA, 1981).
  • These media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • Very good sources of carbon are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugar can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add mixtures of different carbon sources.
  • Other possible sources of carbon are oils and fats such. As soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such. As palmitic acid, stearic acid or linoleic acid, alcohols such. As glycerol, methanol or ethanol and organic acids such. As acetic acid or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
  • Exemplary nitrogen sources include ammonia gas or ammonia water or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium carbamate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used individually or as a mixture.
  • Inorganic salt compounds that may be included in the media include the chloride, phosphate, carbonate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron, and boric acid.
  • sulfur source inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic sulfur compounds, such as mercaptans and thiols can be used.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
  • Chelating agents can be added to the medium to keep the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
  • the culture media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
  • growth factors and salts often originate from complex media components, such as yeast extract, molasses, corn steep liquor and the same.
  • suitable precursors can be added to the culture medium.
  • the exact composition of the media compounds will depend heavily on the particular experiment and will be decided on a case by case basis. Information about the media optimization is available from the textbook "Applied Microbiol. Physiology, A Practical Apache” (ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).
  • Growth media may also be obtained from commercial suppliers such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
  • All media components are sterilized either by heat (eg 20 min at 1 bar overpressure (2 bar absolute) and 121 ° C.) or by sterile filtration.
  • the components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or optionally added continuously or in portions.
  • the temperature of the culture is usually between 15 ° C and 45 ° C, preferably at 25 ° C to 40 0 C and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range of 5 to 8.5, preferably around 7.0.
  • the pH for the fermentation can be controlled during the fermentation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, ammonia or ammonia water or acidic compounds such as phosphoric acid, hydrochloric acid or sulfuric acid.
  • basic compounds such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, ammonia or ammonia water or acidic compounds such as phosphoric acid, hydrochloric acid or sulfuric acid.
  • acidic compounds such as phosphoric acid, hydrochloric acid or sulfuric acid.
  • acidic compounds such as phosphoric acid, hydrochloric acid or sulfuric acid.
  • acidic compounds such as phosphoric acid, hydrochloric acid or sulfuric acid.
  • the medium can be selected selectively acting substances such. As antibiotics, are added.
  • oxygen or oxygen-containing gas mixtures such. B. ambient air, registered in the culture.
  • the temperature of the culture is usually from 20 0 C to 45 ° C.
  • the culture is continued until a maximum of the desired product
  • the fermentation broths thus obtained in particular containing L-lysine or DAP, usually have a dry matter content of from 3 to 20% by weight.
  • the fermentation is driven sugar-limited at least at the end, but especially over at least 30% of the fermentation period. This means that during this time the concentration of utilizable sugar in the fermentation medium is kept at> 0 to 3 g / l, or lowered.
  • the fermentation broth is then further processed.
  • the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation, flocculation or a combination of these methods are removed from the fermentation broth or completely left in her. A separation of the biomass is preferred.
  • the fermentation broth with known methods, such as. B. with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or aufkon- be centered.
  • any salts precipitated by the concentration may be separated by, for example, filtration or centrifugation.
  • This concentrated fermentation broth can then be worked up in the manner according to the invention to obtain DAP.
  • a concentration is possible, but not absolutely necessary.
  • DAP is extracted from the fermentation broth using an organic extractant.
  • an organic solvent having a miscibility gap with water which is as polar as possible and stable in the alkaline, is used, in particular a polar, in particular dipolar protic, organic solvent.
  • Suitable solvents are, in particular, cyclic or open-chain, optionally branched alkanols having 3 to 8 C atoms, such as, in particular, n- and iso-propanol, n-, sec- and isobutanol, or cyclohexanol, and also n-pentanol, n - Hexanol n-heptanol, n-octanol, 2-octanol and the one or more branched isomeric forms thereof.
  • N-butanol is especially worth mentioning.
  • the extraction and / or the subsequent phase separation is carried out batchwise at elevated temperature, the temperature being limited by the boiling points of water and of the extractant or possibly forming azeotropes.
  • n-butanol as the extraction agent extraction and phase separation, for example, at about 25-90 0 C or preferably at 40-70 0 C could be performed.
  • the two phases are stirred until the distribution equilibrium has settled, for example over a period of 10 seconds to 2 hours, preferably 5 to 15 minutes.
  • the phases are allowed to settle until the phases have completely separated; This is preferably carried out over a period of 10 seconds to 5 hours, such as 15 to 120 or 30 to 90 minutes, especially at a temperature in the range of about 25-90 0 C or 40-70 0 C in the case of n-butanol ,
  • the extraction of the DAP from the fermentation broth is carried out continuously in multiple stages (e.g., in mixer-settler combinations) or continuously in an extraction column.
  • the apparatus configuration of the extraction columns which can be used according to the invention can be determined by the person skilled in the art for the phases to be separated in each case in a routine manner. constant optimization work. Suitable are in principle extraction columns without power input or extraction columns with power input, such. B. pulsed columns or columns with rotating internals. The person skilled in the art can also select the type and materials of internals, such as sieve trays, and column packings in the course of routine work, in order to optimize the phase separation in a suitable manner.
  • the theoretical foundations of liquid-liquid extraction of small molecules are well known (see, for example, H.-J. Rehm and G. Reed, Eds., (1993), Biotechology, Volume 3 Bioprocessing, Chapter 21, VCH, Weinheim). The design of industrially applicable extraction columns is described, for example, in Lo et al., Eds., (1983) Handbook of Solvent Extraction, John Wiley & Sons, New York. The disclosure of the above textbooks is expressly incorporated by reference.
  • the isolation and purification of the DAP from the DAP-containing extract phase are carried out in a manner known per se.
  • Possible measures for DAP recovery include, but are not limited to, distillation, precipitation as a salt with suitable organic or inorganic acids, or combinations of such suitable measures.
  • the distillation can be carried out continuously or batchwise (batchwise).
  • a single or more coupled distillation columns can be used.
  • the apparatus design of the distillation column and the determination of the operating parameters is the expert.
  • the distillation columns used in each case can be realized in a manner known per se (see, for example, Sattler, Thermal Separation Methods, 2nd Edition 1995, Weinheim, page 135 et seq., Perry's Chemical Engineers Handbook, 7th Edition 1997, New York, Section 13 ).
  • the distillation columns used may be e.g. separating internals included, such as separating trays, z. As perforated plates, bubble trays or valve trays, ordered packs, z. As sheet or tissue packs, or random beds of packing.
  • the number of stages and the reflux ratio necessary in the column (s) used depend essentially on the purity requirements and the relative boiling position of the liquids to be separated, with the person skilled in the art being able to determine the specific design and operating data by known methods.
  • the precipitation as a salt can be brought about by addition of suitable organic or inorganic acids, such as, for example, sulfuric acid, hydrochloric acid, phosphoric acid. acid, acetic acid, formic acid, carbonic acid, oxalic acid, etc.
  • suitable organic or inorganic acids such as, for example, sulfuric acid, hydrochloric acid, phosphoric acid. acid, acetic acid, formic acid, carbonic acid, oxalic acid, etc.
  • an organic dicarboxylic acid is used which leads to the formation of a salt which can be used directly or after purification, for example by recrystallization, in a subsequent polycondensation to the polyamide.
  • dicarboxylic acids may be C 4 -C 2 dicarboxylic acids in particular.
  • the organic DAP phase obtained during the extraction can also be worked up by chromatography.
  • the DAP phase is applied to a suitable resin, e.g. a strong or weakly acidic ion exchanger (such as Lewatit 1468 S, Dowex Marathon C, Amberlyst 1 19 Wet or others) with the desired product or impurities retained in whole or in part on the chromatography resin. If necessary, these chromatographic steps can be repeated using the same or different chromatography resins.
  • a strong or weakly acidic ion exchanger such as Lewatit 1468 S, Dowex Marathon C, Amberlyst 1 19 Wet or others
  • the purified product may be concentrated by filtration or ultrafiltration and stored at a suitable temperature.
  • the identity and purity of the isolated compound (s) can be determined by techniques of the prior art. These include high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopic methods, staining procedures, thin layer chromatography, NIRS, enzyme assay or microbiological assays. These analytical methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 1 1 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp.
  • the cells were inoculated with a loop from the Petri dish (culture 2) in 200 ml of a 2L shake flask with 2 baffles (composition analog plate and batch medium) and at 30 0 C on an orbital shaker at a speed of 250 rpm for Incubated for 24 h.
  • the contents of the shake flasks served as a preculture for the inoculation of a 75 L fermenter with a filling volume of 50 L.
  • the pH was controlled with the aid of ammonia gas to pH 6.8.
  • the fumigation rate was about 0.33 vvm.
  • the main culture was carried out in a 5 m 3 kettle with a 700 L filling volume in the batch phase. After a further 24 h, the culture was transferred from the 75 L fermenter to the 5 m 3 kettle. The pH was adjusted to pH 6.8 with ammonia. The dissolved oxygen was controlled in the range of 20 to 30% (air saturation) by adjusting the gasification rate and stirrer speed.
  • the glucose concentration of the batch medium dropped to a value below 1 g / L and the feed was started.
  • the final volume of the fermentation of about 3200 L was reached.
  • the final concentrations in the fermentation were OD 6 io: 140, 1, 5-diaminopentane: 72 g / L, lysine x HCl: 15 g / L, acetyldiaminopentane: 10 g / L.
  • Embodiment 1 Recovery of DAP from fermentation broth of a lysine and DAP producing microorganism
  • Test Example 1 Investigation of the rate of mass transfer in the extraction
  • the rate of phase separation was investigated in a 2.5 L jacketed reactor with 3-stage bar stirrer and 4 baffles.
  • Table 3 Phase separation times on extraction of a broth with yeast extract without thermal treatment
  • the production organism additionally acetylates a part of the DAP formed on one of the two amino groups.
  • acetyl-diaminopentane can be saponified by refluxing the fermentation broth, which has been made alkaline to a pH above 13, with the release of the diaminopentane (see Fig. 2). This can increase the yield. Saponification under acidic conditions (pH 1 with H 2 SO 4 ) is very slow.
  • a pH of at least 13.5 is advantageous for successful boiling.

Abstract

The invention relates to a method for isolating 1,5-diaminopentane (DAP) from DAP-containing fermentation broths, to a method for the fermentative production of DAP using said isolating method and to a method for producing DAP-comprising polymers by using the DAP isolated or fermatively produced DAPs according to said methods.

Description

Verfahren zur fermentativen Herstellung von 1,5-Diaminopentan Process for the fermentative production of 1,5-diaminopentane
Die vorliegende Erfindung betrifft ein Verfahren zur Isolierung von 1 ,5-Diaminopentan (DAP) aus DAP-haltigen Fermentationsbrühen, ein Verfahren zur fermentativen Her- Stellung von DAP unter Verwendung dieser Isolierungsmethode sowie ein Verfahren zur Herstellung von DAP-haltigen Polymeren unter Einsatz des in dieser Weise isolierten bzw. fermentativ hergestellten DAPs.The present invention relates to a process for the isolation of 1, 5-diaminopentane (DAP) from DAP-containing fermentation broths, a process for the fermentative production of DAP using this isolation method and a process for the preparation of DAP-containing polymers using the in this way isolated or fermentatively produced DAPs.
HINTERGRUND DER ERFINDUNGBACKGROUND OF THE INVENTION
1 ,5-Diaminopentan (häufig auch bezeichnet als Pentamethylendiamin oder Cadaverin; im Folgenden bezeichnet als DAP) stellt einen wichtigen Grundstoff der chemischen Industrie dar. Beispielsweise findet DAP Verwendung bei der Herstellung von Polyamiden, Polyharnstoffen oder Polyurethanen sowie von Copolymeren daraus.1,5-Diaminopentane (often also referred to as pentamethylenediamine or cadaverine, hereinafter referred to as DAP) represents an important raw material of the chemical industry. For example, DAP is used in the production of polyamides, polyureas or polyurethanes and copolymers thereof.
Zudem ist die fermentative oder enzymatische Herstellung von DAP durch Decarboxy- lierung von Lysin seit längerem bekannt. Dabei werden verschiedene Verfahren zur Isolierung des Wertproduktes aus der Fermentationsbrühe beschrieben.In addition, the fermentative or enzymatic production of DAP by decarboxylation of lysine has long been known. Various methods for isolating the desired product from the fermentation broth are described.
So wird beispielsweise in der EP-A-1 482 055 die enzymatische Decarboxylierung von Lysin in Gegenwart einer Dicarbonsäure zur Einstellung des pH-Wertes während der Umsetzung beschrieben. Das bei der Herstellung anfallende DAP-Dicarboxylat wird isoliert, indem man die wertstoffhaltige Lösung zunächst mit Aktivkohle entfärbt, aufkonzentriert und DAP-Dicarboxylat durch eine Kühlungskristallisation auskristallisiert.Thus, for example, EP-A-1 482 055 describes the enzymatic decarboxylation of lysine in the presence of a dicarboxylic acid for adjusting the pH during the reaction. The DAP dicarboxylate obtained in the preparation is isolated by first destaining the concentrated solution with activated charcoal, concentrating and crystallizing out DAP dicarboxylate by cooling crystallization.
In der WO-A-2006/123778 ist die Herstellung von DAP-Carbonat durch enzymatische Decarboxylierung von Lysin in Gegenwart von Kohlendioxid beschrieben. Durch Aufkonzentrierung der Reaktionslösung und Abspaltung von Kohlendioxid wird DAP gebildet.WO-A-2006/123778 describes the preparation of DAP carbonate by enzymatic decarboxylation of lysine in the presence of carbon dioxide. Concentration of the reaction solution and removal of carbon dioxide DAP is formed.
Die JP 2004-208 646 beschreibt die Herstellung von DAP-Dicarboxylat durch enzymatische Decarboxylierung einer L-Lysin-Dicarboxylat enthaltenden Lösung und Ausfällung von DAP-Dicarboxylat durch Zugabe eines organischen Lösungsmittels, ausgewählt unter Alkoholen, Ketonen und Nitrilen.JP 2004-208 646 describes the preparation of DAP dicarboxylate by enzymatic decarboxylation of a solution containing L-lysine dicarboxylate and precipitation of DAP dicarboxylate by addition of an organic solvent selected from among alcohols, ketones and nitriles.
Die JP 2004-222 569 beschreibt die Herstellung von DAP unter Verwendung eines L- Lysin-Decarboxylase-exprimierenden coryneformen Bakteriums, Einstellung des KuI- turüberstandes auf pH 12 und Extraktion von DAP mit einem polaren organischen Lösungsmittel.JP 2004-222 569 describes the production of DAP using an L-lysine decarboxylase-expressing coryneform bacterium, setting the KuI supernatant to pH 12 and extraction of DAP with a polar organic solvent.
Schließlich beschreibt die JP 2004-000 114 die Herstellung von DAP durch Umsetzung von hochkonzentriertem L-Lysin-Monohydrochlorid mit L-Lysin-Decarboxylase- exprimierenden E. co//-Zellen, Einstellung der Reaktionslösung auf pH ≥13 und Extraktion des Reaktionsproduktes mit einem polaren organischen Lösungsmittel und anschließender Destillation.Finally, JP 2004-000114 describes the preparation of DAP by reacting highly concentrated L-lysine monohydrochloride with L-lysine decarboxylase-expressing E. coli cells, adjusting the reaction solution to pH ≥13 and extracting the reaction product with one polar organic solvent and subsequent distillation.
Insbesondere die aus dem Stand der Technik bekannten, auf einer Extraktion von DAP mit Hilfe eines organischen Lösungsmittels basierenden Verfahren sind jedoch mit dem Nachteil behaftet, dass die Wertstoffausbeute nicht optimal ist und insbesondere der Extraktionsschritt zu langsam verläuft und das Gesamtverfahren daher zu zeitaufwän- dig ist, was für eine Anwendung der Herstellung im technischen Maßstab von großem Nachteil ist.However, in particular the processes known from the prior art, based on extraction of DAP with the aid of an organic solvent, have the disadvantage that the recycling of the valuable material is not optimal and in particular the extraction step is too slow and the overall process is therefore too time-consuming , which is a great disadvantage for an application of the production on an industrial scale.
ZUSAMMENFASSUNG DER ERFINDUNGSUMMARY OF THE INVENTION
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, die Isolierung von DAP (Cadaverin) aus Fermentationsbrühen weiter zu verbessern. Insbesondere sollte die Ausbeute an Wertstoff weiter erhöht und der erforderliche Zeitaufwand für die Isolierung, insbesondere die Lösungsmittel-basierte Extraktion, verbessert werden.The present invention is therefore based on the object of further improving the isolation of DAP (cadaverine) from fermentation broths. In particular, the yield of valuable material should be further increased and the time required for the isolation, in particular the solvent-based extraction, improved.
Überraschenderweise wurde diese Aufgabe durch Bereitstellung eines Verfahrens ge- löst, bei welchem man die auf einen alkalischen pH-Wert eingestellte Fermentationsbrühe thermisch behandelt und anschließend mit einem geeigneten organischen Extraktionsmittel extrahiert. Dabei wurde überraschenderweise festgestellt, dass DAP- haltige Nebenprodukte der Fermentation, insbesondere Acetyl-DAP unter Freisetzung des Wertproduktes hydrolytisch gespalten werden und überraschenderweise außer- dem die Geschwindigkeit der Phasentrennung während des Extraktionsschrittes deutlich erhöht werden kann. Die erhöhte Geschwindigkeit der Phasentrennung ist besonders evident bei der Aufarbeitung von Fermentationsbrühen aus der Fermentation von Mikroorganismen in Gegenwart von komplexen Nährmedien, wie z.B. Hefeextrakt. FIGURENBESCHREIBUNGSurprisingly, this object has been achieved by providing a process in which the treated to an alkaline pH fermentation broth is thermally treated and then extracted with a suitable organic extractant. It was surprisingly found that DAP-containing by-products of the fermentation, especially acetyl-DAP are hydrolytically cleaved to release the desired product and, surprisingly, the rate of phase separation during the extraction step can be significantly increased. The increased rate of phase separation is particularly evident in the processing of fermentation broths from the fermentation of microorganisms in the presence of complex nutrient media, such as yeast extract. DESCRIPTION OF THE FIGURES
Figur 1 zeigt ein Blockschema für den Verlauf einer speziellen erfindungsgemäßen Ausführungsform eines Gesamtprozesses zur Isolierung von DAP aus einer Fermenta- tionsbrühe.FIG. 1 shows a block diagram for the course of a specific embodiment according to the invention of an overall process for isolating DAP from a fermentation broth.
Figur 2 veranschaulicht die hydrolytische Spaltung von Acetyl-DAP (Dreiecke) unter Bildung von DAP (Rauten) im Verlauf einer fünfstündigen thermischen Behandlung einer Fermentationsbrühe durch Rückflusskochen bei pH 13,7. Der Gehalt an restli- ehern Lysin (Quadrate) bleibt während der thermischen Behandlung unverändert.Figure 2 illustrates the hydrolytic cleavage of acetyl-DAP (triangles) to form DAP (diamonds) over a five hour thermal treatment of a fermentation broth by refluxing at pH 13.7. The content of residual lysine (squares) remains unchanged during the thermal treatment.
Figur 3 veranschaulicht die Freisetzung von Ammoniak während des Aufheizvorgangs und des anschließenden Rückflusskochens der Fermentationsbrühe. Untere Kurve - Gasmenge; obere Kurve - Innentemperaturverlauf.Figure 3 illustrates the release of ammonia during the heating process and the subsequent refluxing of the fermentation broth. Lower curve - gas quantity; upper curve - internal temperature profile.
DETAILLIERTE BESCHREIBUNG DER ERFINDUNGDETAILED DESCRIPTION OF THE INVENTION
1. Bevorzugte Ausführungsformen1. Preferred embodiments
Gegenstand der Erfindung ist ein Verfahren zur Isolierung von 1 ,5-Diaminopentan (DAP) aus einer DAP-haltigen Fermentationsbrühe, wobei man die Fermentationsbrühe a) alkalisiert, b) thermisch behandelt, c) DAP mit einem organischen Extraktionsmittel extrahiert, und d) DAP aus der abgetrennten organischen Phase isoliert.The invention relates to a method for the isolation of 1, 5-diaminopentane (DAP) from a DAP-containing fermentation broth, wherein the fermentation broth a) alkalized, b) thermally treated, c) extracted DAP with an organic extractant, and d) DAP isolated from the separated organic phase.
In einer ersten speziellen Ausgestaltung des Verfahrens wird die Fermentationsbrühe auf einen pH-Wert von >1 1 , wie insbesondere ≥1 1 ,5 oder ≥12, wie insbesondere ≥12 bis 14, oder 12,5 bis 13,8, oder 13 bis 13,8, oder 13,5 bis 13,7 eingestellt. Dazu erfolgt die pH-Wert-Einstellung insbesondere durch Zugabe eines Alkali- oder Erdalkalimetallhydroxids, wie eines Na-, K- oder Ca-Hydroxids.In a first specific embodiment of the method, the fermentation broth to a pH of> 1 1, in particular ≥1 1, 5 or ≥12, in particular ≥12 to 14, or 12.5 to 13.8, or 13 to 13.8, or 13.5 to 13.7. For this purpose, the pH adjustment is carried out in particular by adding an alkali metal or alkaline earth metal hydroxide, such as a Na, K or Ca hydroxide.
Die Stoffverteilung kann durch Anpassung des pH Wertes weiter optimiert werden, wobei bei pH-Werten oberhalb von etwa 12,5 optimale Stoffübergangsbedingungen eingestellt können.The material distribution can be further optimized by adjusting the pH, with optimum mass transfer conditions being able to be set at pH values above about 12.5.
Ebenso kann die Spaltung von gegebenenfalls enthaltenem Acetyl-DAP durch Anpassung des pH-Wertes weiter optimiert werden, wobei - abhängig von der Menge an enthaltenem Acetyl-DAP - bei pH-Werten oberhalb von etwa 13 optimale Spaltungsbedingungen (Spaltungskinetiken) eingestellt werden können.Likewise, the cleavage of optionally contained acetyl-DAP can be further optimized by adjusting the pH, wherein - depending on the amount of contained acetyl-DAP - can be adjusted at pH values above about 13 optimal cleavage conditions (cleavage kinetics).
Gegebenenfalls können vor der Alkalisierung aus der Fermentationsbrühe zelluläre Bestandteile entfernt werden. Methoden zur Entfernung der zellulären Bestandteile sind dem Fachmann geläufig (z.B. Separatoren, Dekanter, Flockung, Filtrationsverfahren, bzw. Kombinationen mehrerer solcher Prozessschritte).Optionally, cellular components may be removed from the fermentation broth prior to alkalinization. Methods for removing the cellular components are well known to those skilled in the art (e.g., separators, decanters, flocculation, filtration methods, or combinations of several such process steps).
In einer weiteren Ausgestaltung des Verfahrens wird die alkalisierte Fermentationsbrü- he thermisch behandelt, indem man, z.B. Batch-weise oder kontinuierlich, auf Rückflusstemperatur, wie z.B. 90-1 10 0C bei Normaldruck oder auf eine höhere Temperatur bei Überdruck, wie z.B. 0-100 bar, insbesondere 0-25 bar Überdruck, erhitzt. Dabei wird die thermische Behandlung unter Bedingungen durchgeführt, die eine, bevorzugt im wesentlichen quantitative, hydrolytische Spaltung von gegebenenfalls vorhandenem Acetyl-DAP bewirken. Der Fachmann kann dazu die wesentlichen Verfahrensparameter, wie Druck, Temperatur und Verweilzeit dem Erfordernis entsprechend aufeinander abstimmen. Der Begriff „Acetyl-DAP" umfasst Mono- und Di-Acetyl-DAP, wobei gewöhnlich aber überwiegend die Mono-Acetylform vorliegt. In einer weiteren Ausführungsform kann diese Erhitzung mehrstufig durchgeführt werden, z.B. auch unter Wie- dergewinnung des freigesetzten Ammoniaks durch Zwischenentspannung.In a further embodiment of the process, the alkalized fermentation broth is thermally treated by passing, for example batchwise or continuously, to reflux temperature, such as 90-1 10 0 C at atmospheric pressure or to a higher temperature at overpressure, such as 0- 100 bar, in particular 0-25 bar overpressure, heated. The thermal treatment is carried out under conditions which cause a, preferably substantially quantitative, hydrolytic cleavage of optionally present acetyl-DAP. The person skilled in the art can coordinate the essential process parameters, such as pressure, temperature and residence time, as required. The term "acetyl-DAP" includes mono- and di-acetyl-DAP, but usually the mono-acetyl form is predominantly present In a further embodiment, this heating can be carried out in several stages, eg also with recovery of the liberated ammonia by intermediate relaxation.
In einer weiteren Ausgestaltung des erfindungsgemäßen Verfahrens wird die DAP- Extraktion mit einem organischen Lösungsmittel mit Mischungslücke mit Wasser, das möglichst polar und im Alkalischen stabil ist, wie insbesondere einem polaren, insbe- sondere dipolar-protischen, organischen Lösungsmittel durchgeführt. Geeignete Lösungsmittel werden in einem folgenden Abschnitt beschriebenIn a further embodiment of the process according to the invention, the DAP extraction is carried out with an organic solvent with miscibility gap with water which is as polar as possible and stable in the alkaline, in particular a polar, in particular dipolar protic, organic solvent. Suitable solvents are described in a following section
In einer bevorzugten Ausführungsform wird die DAP-Extraktion und/oder die anschließende Phasentrennung diskontinuierlich bei erhöhter Temperatur durchgeführt. Weite- re Ausgestaltungen der Extraktion und der Aufarbeitung des DAP-haltigen Extraktes werden in einem folgenden Abschnitt beschrieben.In a preferred embodiment, the DAP extraction and / or the subsequent phase separation is carried out batchwise at elevated temperature. Further embodiments of the extraction and the workup of the DAP-containing extract are described in a following section.
Insbesondere eignet sich das erfindungsgemäße Verfahren für die Aufarbeitung von Fermentationsbrühen aus der Fermentation eines Mikroorganismus in einem komple- xen, wie z.B. Hefeextrakt-haltigen, Kulturmedium. Ein weiterer Gegenstand der Erfindung ist ein Verfahren zur fermentativen Herstellung von DAP, wobei man einen Lysin-produzierenden Mikroorganismus unter Lysin- und gegebenenfalls DAP-produzierenden Bedingungen kultiviert und das gebildete DAP unter Anwendung eines DAP-Isolationsverfahrens nach obiger Definition isoliert.In particular, the process according to the invention is suitable for the processing of fermentation broths from the fermentation of a microorganism in a complex culture medium, such as, for example, yeast extract-containing culture medium. Another object of the invention is a process for the fermentative production of DAP, wherein a lysine-producing microorganism is cultured under lysine and optionally DAP-producing conditions and the resulting DAP isolated using a DAP isolation method as defined above.
Insbesondere kann dabei die Fermentation in einem Kulturmedium mit komplexen Medienbestandteilen durchgeführt werden. Dabei kann man einen Lysin-produzierenden Mikroorganismus verwenden, der zusätzlich Lysin Decarboxylase-Aktivität, wie z.B. eine heterologe, d.h. aus einem anderen Organismus stammende, Lysin Decarboxyla- se (LDC), exprimiert.In particular, the fermentation can be carried out in a culture medium with complex media components. In this case one can use a lysine-producing microorganism, which additionally lysine decarboxylase activity, such as. a heterologous, i. from another organism, lysine decarboxylase (LDC) expressed.
„Komplexe Nähr- oder Kulturmedien" oder „Komplexmedien" sind erfindungsgemäß an sich bekannte Medien, welche komplex zusammengesetzte Stoffgemische umfassen, wie z.B. Maisquellwasser, Trypton, Bacton, Sojahydrolysat und insbesondere Hefeex- trakt."Complex nutrient or culture media" or "complex media" are, according to the invention, media known per se which comprise mixtures of substances of complex composition, such as e.g. Corn steep liquor, tryptone, bactone, soy hydrolyzate and, in particular, yeast extract.
Andererseits kann auch eine Lysin-haltige Fermentationsbrühe mit gereinigter, gegebenenfalls immobilisierter Lysin-Decarboxylase in Kontakt gebracht werden, um Lysin zu DAP zu decarboxylieren oder ein weiterer, gegebenenfalls immobilisierter LDC- exprimierender Mikroorganismus kann der Brühe zugesetzt oder diese damit in Kontakt gebracht werden. Geeignete Verfahren sind dabei im Stand der Technik beschrieben, worauf hiermit ausdrücklich Bezug genommen wird. (vgl. z.B. JP 2002-223771 ).Alternatively, a lysine-containing fermentation broth may be contacted with purified, optionally immobilized lysine decarboxylase to decarboxylate lysine to DAP, or another optionally immobilized LDC-expressing microorganism may be added to or contacted with the broth. Suitable processes are described in the prior art, to which reference is hereby expressly made. (See, for example, JP 2002-223771).
Grundsätzlich kann dabei das gesamte oben beschriebene Isolierungsverfahren bzw. das oben beschriebene fermentative Herstellungsverfahren oder einzelne Schritte davon kontinuierlich oder diskontinuierlich, in Batch- oder Semibatch-, oder Fed-Batch- oder repeated (Fed-)Batch-Fahrweise, erfolgen.In principle, the entire isolation process described above or the fermentative preparation process described above or individual steps thereof can be carried out continuously or discontinuously, in batch or semibatch, or fed-batch or repeated (fed) batch mode.
Ein weiterer Gegenstand der Erfindung betrifft ein Verfahren zur Herstellung eines DAP-haltigen Polymers, wobei man zunächst monomeres DAP nach einem Verfahren gemäß obiger Definition fermentativ herstellt und isoliert und zusammen mit wenigsten einem weiteren Comonomer polymerisiert. Dabei kann das Comonomer insbesondere ausgewählt sein unter Polycarbonsäuren, wie insbesondere Dicarbonsäuren mit 4 bisAnother object of the invention relates to a process for the preparation of a DAP-containing polymer, wherein first prepared monomeric DAP by a process according to the above definition by fermentation and isolated and polymerized together with at least one other comonomer. In this case, the comonomer may in particular be selected from polycarboxylic acids, in particular dicarboxylic acids with 4 to
12 Kohlenstoffatomen, deren Estern und Anhydriden; sowie Polyisocyanaten, wie ins- besondere Diisocyanaten mit einer C2-Ci0-Alkylen-Brückengruppe oder cyclischen12 carbon atoms, their esters and anhydrides; and polyisocyanates, in particular diisocyanates, such as with a C 2 -C 0 -alkylene bridge group or cyclic
Brückengruppen. Nichtlimitierende Beispiele geeigneter Dicarbonsäuren sind Bern- steinsäure, Glutarsäure, Adipinsäure, Pimelinsäure, Suberinsäure, Azelainsäure, Se- bacinsäure usw. Nichtlimitierende Beispiele geeigneter Diisocyanate sind Methylen- diphenyldiisocyanat (MDI), Toluoldiisocyanat (TDI), Hexamethylendiisocyanat (HDI) und Isophorondiisocyanat. Dabei werden insbesondere Polymere vom Polyamid-, Po- lyharnstoff- oder Polyurethan-Typ gebildet, wie z.B. Polyamid 5,10 oder Polyamid 5,6.Bridge groups. Nonlimiting examples of suitable dicarboxylic acids are Bern nonlimiting examples of suitable diisocyanates are methylene diphenyl diisocyanate (MDI), toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI) and isophorone diisocyanate. In particular, polymers of the polyamide, polyurea or polyurethane type are formed, for example polyamide 5, 10 or polyamide 5, 6.
Für das Polymerisationsverfahren setzt man wenigstens ein Comonomer dem isolierten DAP zu oder setzt ein Gemisch aus DAP und wenigstens einem Comonomer aus einer DAP-Fällung ein. Beispielsweise kann ein geeignetes DAP/Comonomer-Gemisch aus einer oben beschriebenen Salzfällung von DAP aus einem destillativ aufgearbeiteten DAP-Extrakt resultieren. Das Comonomer ist dabei vorzugsweise eine Polycarbon- säure, wie beispielsweise Sebacinsäure.For the polymerization process, at least one comonomer is added to the isolated DAP or a mixture of DAP and at least one comonomer from a DAP precipitation is used. For example, a suitable DAP / comonomer mixture from a salt precipitation of DAP described above may result from a distilled reclaimed DAP extract. The comonomer is preferably a polycarboxylic acid, such as sebacic acid.
2. Allgemeine Angaben zur fermentativen Lvsin- oder DAP-Herstellung2. General information on the fermentative production of Lvsin or DAP
2.1 Mikroorganismen2.1 microorganisms
Die vorliegende Erfindung ist grundsätzlich auf die Aufarbeitung jeglicher DAP-haltiger Fermentationsbrühen anwendbar. Auch bezüglich der bei der Fermentation verwende- ten Mikroorganismen bestehen grundsätzlich keinerlei Beschränkungen. Dabei kann es sich um natürlich vorkommende, um durch Mutation und Selektion verbesserte, insbesondere aber um rekombinant hergestellte Mikroorganismen, wie Pilze, insbesondere aber Bakterien handeln. Diese Mikroorganismen besitzen entweder direkt die Fähigkeit zur Produktion von DAP und/oder DAP-Derivaten, wie Acetyl-DAP, zumindest aber sind sie zur fermentativen Herstellung von Lysin, insbesondere L-Lysin befähigt. Insbesondere ist ein verwendetes rekombinantes Bakterium zur Lysin-Biosynthese über den Diaminopimelat-Weg („DAP-Weg"), den Succinylase-Weg oder den Dehydrogenase- Weg befähigt.The present invention is basically applicable to the processing of any DAP-containing fermentation broths. In principle, there are also no restrictions with regard to the microorganisms used in the fermentation. These may be naturally occurring, improved by mutation and selection, but especially recombinant microorganisms such as fungi, but especially bacteria. These microorganisms have either the direct ability to produce DAP and / or DAP derivatives, such as acetyl-DAP, but at least they are capable of fermentative production of lysine, especially L-lysine. In particular, a recombinant bacterium used for lysine biosynthesis via the diaminopimelate pathway ("DAP pathway"), the succinylase pathway or the dehydrogenase pathway is capable.
Diese Mikroorganismen können Lysin, insbesondere L-Lysin, aus Glucose, Saccharose, Lactose, Fructose, Maltose, Melasse, Stärke, Cellulose oder aus Glycerin, Fettsäuren oder Pflanzenölen oder Ethanol produzieren und vorzugsweise das gebildete Lysin zumindest teilweise in den extrazellulären Raum abgeben. Vorzugsweise sind dies coryneforme Bakterien, insbesondere der Gattung Corynebacterium oder der Gattung Brevibacterium. Aus der Gattung Corynebacterium ist insbesondere die Art Corynebac- terium glutamicum zu nennen, die in der Fachwelt für ihre Fähigkeit bekannt ist, L- Aminosäuren zu produzieren.These microorganisms can produce lysine, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol, fatty acids or vegetable oils or ethanol and preferably at least partially release the lysine formed into the extracellular space. Preferably, these are coryneform bacteria, in particular the genus Corynebacterium or the genus Brevibacterium. From the genus Corynebacterium in particular the species Corynebac- terium glutamicum, which is known in the art for its ability to produce L-amino acids.
Als Beispiele für geeignete Stämme coryneformer Bakterien sind solche der Gattung Corynebacterium, insbesondere der Art Corynebacteήum glutamicum (C. glutamicum), wieExamples of suitable strains of coryneform bacteria are those of the genus Corynebacterium, in particular of the species Corynebacteήum glutamicum (C. glutamicum), such as
Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium acetoacidophilum ATCC 13870,Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium acetoacidophilum ATCC 13870,
Corynebacterium thermoaminogenes FERM BP-1539, Corynebacterium melassecola ATCC 17965Corynebacterium thermoaminogenes FERM BP-1539, Corynebacterium molassecola ATCC 17965
oder der Gattung Brevibacterium, wieor the genus Brevibacterium, such as
Brevibacterium flavum ATCC 14067 Brevibacterium lactofermentum ATCC 13869 und Brevibacterium divaricatum ATCC 14020 zu nennen;Brevibacterium flavum ATCC 14067 Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC 14020;
oder davon abgeleitete Stämme, wieor derived strains, such as
Corynebacterium glutamicum KFCC10065 Corynebacterium glutamicum ATCC21608Corynebacterium glutamicum KFCC10065 Corynebacterium glutamicum ATCC21608
Mit der Abkürzung KFCC ist die Korean Federation of Culture Collection gemeint, mit der Abkürzung ATCC die American type strain culture collection, mit der Abkürzung FERM BP die Sammlung des National institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan bezeichnet.The abbreviation KFCC means the Korean Federation of Culture Collection, the abbreviation ATCC denotes the American strain strain culture collection, abbreviated FERM BP the collection of the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan ,
2.2 Durchführung der Fermentation2.2 Carrying out the fermentation
Erfindungsgemäß aufzuarbeitende Fermentationsbrühen stammen beispielsweise aus der Kultivierung von rekombinanten coryneformen Bakterien, welche über einen die Lysin-Biosynthese fördernden, wenigstens ein Lysinbiosynthese-Gen betreffenden deregulierenden Eingriff, vermehrt Lysin, insbesondere L-Lysin, oder ein Lysin-haltiges Stoffgemisch produzieren und / oder welche zusätzlich ein Enzym mit Lysin Decarbo- xylase-Aktivität überexprimieren und DAP und/oder Acetyl-DAP akkumulieren. Letztere sind damit zur direkten DAP-Produktion befähigt.Fermentation broths to be worked up according to the invention originate, for example, from the cultivation of recombinant coryneform bacteria, which produce a lysin biosynthesis-promoting deregulatory intervention involving at least one lysine biosynthesis gene, lysine, in particular L-lysine or a lysine-containing substance mixture and / or which additionally an enzyme with lysine decarbo- overexpressing xylase activity and accumulating DAP and / or acetyl-DAP. The latter are thus capable of direct DAP production.
An der Lysin-Biosynthese beteiligte Gene und ein zugeordneter, die Lysin-Biosynthese fördernder deregulativer Eingriff sind in folgender Tabellei zusammengefasst.Genes involved in lysine biosynthesis and associated lysine biosynthesis-promoting deregulation are summarized in the following table.
Tabelle 1 : Beispiele deregulierbarer Gene und GenprodukteTable 1: Examples of deregulatory genes and gene products
Ein Verfahren zur Herstellung von DAP unter Verwendung rekombinanter Mikroorganismen mit dereguliertem Lysin-Decarboxylasegen und wenigstens einem weiteren deregulierten, z.B. an der Lysinbiosynthese beteiligten Gen, ist aus der WO 2007/113127 bekannt, worauf hiermit ausdrücklich Bezug genommen wird.A method of producing DAP using recombinant microorganisms with deregulated lysine decarboxylase gene and at least one further deregulated, e.g. The gene involved in lysine biosynthesis is known from WO 2007/113127, to which reference is hereby expressly made.
Wie ersichtlich, ist eine „Deregulation" im weitesten Sinne zu verstehen und umfasst sowohl eine Erhöhung oder Verringerung oder Abschaltung einer Enzymaktivität in verschiedenster Weise, z.B. durch Erhöhung oder Verringerung der Kopienzahl von Enzymmolekülen im Mikroorganismus oder eine Veränderung einer anderen, die Lysin- Biosynthese verringernden Eigenschaft.As can be seen, "deregulation" is to be understood in the broadest sense and encompasses both increasing or decreasing or shutting down enzyme activity in a variety of ways, eg by increasing or decreasing the copy number of enzyme molecules in the microorganism or changing another one which reduces lysine biosynthesis Property.
Das Enzym Lysin Decarboxylase (E. C. 4.1.1.18.) katalysiert die Decarboxylierung von L-Lysin zu DAP. Das Enzym ist beispielsweise das cadA Genprodukt (Kyoto Encyclo- pedia of Genes and Genomes, Entry b4131 ) oder das IdcC Genprodukt (Kyoto Encyc- lopedia of Genes and Genomes, Entry JW0181 ). Deren Verwendung zur Herstellung rekombinanter Mikroorganismen für die Cadaverin-Produktion sind dem Fachmann bekannt (vgl. z.B. EP-A-1 482 055).The enzyme lysine decarboxylase (E.C. 4.1.1.18.) Catalyzes the decarboxylation of L-lysine to DAP. The enzyme is, for example, the cadA gene product (Kyoto Encyclopedia of Genes and Genomes, Entry b4131) or the IdcC gene product (Kyoto Encyclopedia of Genes and Genomes, Entry JW0181). Their use for the production of recombinant microorganisms for cadaverine production are known to the person skilled in the art (cf., for example, EP-A-1 482 055).
Zur Erzielung einer Überexpression/Deregulierung kann der Fachmann unterschiedliche Maßnahmen einzeln oder in Kombination ergreifen. So kann die Kopienzahl der entsprechenden Gene erhöht werden, oder es kann die Promotor- und Regulationsregion oder die Ribosomenbindungsstelle, die sich stromaufwärts des Strukturgens befindet, mutiert werden. In gleicher Weise wirken Expressionskassetten, die stromauf- wärts des Strukturgens eingebaut werden. Durch induzierbare Promotoren ist es zusätzlich möglich, die Expression im Verlaufe der fermentativen L-Lysin-Produktion zu steigern. Durch Maßnahmen zur Verlängerung der Lebensdauer der mRNA wird ebenfalls die Expression verbessert. Weiterhin wird durch Verhinderung des Abbaus des Enzymproteins ebenfalls die Enzymaktivität verstärkt. Die Gene oder Genkonstrukte können entweder in einem oder mehreren Plasmiden mit unterschiedlicher Kopienzahl vorliegen oder im Chromosom integriert und amplifiziert sein. Alternativ kann weiterhin eine Überexpression der betreffenden Gene durch Veränderung der Medienzusammensetzung und Kulturführung erreicht werden.To achieve overexpression / deregulation, the skilled person can take different measures individually or in combination. Thus, the copy number of the corresponding genes can be increased, or the promoter and regulatory region or ribosome binding site located upstream of the structural gene can be mutated. In the same way, expression cassettes which act upstream be incorporated into the structural gene. By inducible promoters it is additionally possible to increase expression in the course of fermentative L-lysine production. Measures to extend the lifetime of mRNA also improve expression. Furthermore, by preventing degradation of the enzyme protein, enzyme activity is also enhanced. The genes or gene constructs can either be present in one or more plasmids with different copy numbers or be integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can be achieved by changing the composition of the medium and culture.
Anleitungen hierzu findet der Fachmann unter anderem bei Martin et al. (Biotechnology 5, 137-146 (1987)), bei Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya und Mori- naga (Bio/Technology 6, 428-430 (1988)), bei Eikmanns et al. (Gene 102, 93-98 (1991 )), in der Europäischen Patentschrift 0472869, im US Patent 4,601 ,893, bei Schwarzer und Pühler (Biotechnology 9, 84-87 (1991 ), bei Remscheid et al. (Applied and Environmental Microbiology 60,126-132 (1994), bei LaBarre et al. (Journal of Bac- teriology 175, 1001-1007 (1993)), in der Patentanmeldung WO 96/15246, bei Ma- lumbres et al. (Gene 134, 15-24 (1993)), in der japanischen Offenlegungsschrift JP-A- 10-229891 , bei Jensen und Hammer (Biotechnology and Bioengineering 58, .191-195 (1998)), bei Makrides (Microbiological Reviews 60 : 512-538 (1996) und in bekannten Lehrbüchern der Genetik und Molekularbiologie.For instructions on this, the skilled person will find, inter alia, in Martin et al. (Biotechnology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), European Patent 0472869, U.S. Patent 4,601,893, Schwarzer and Pühler (Biotechnology 9, 84-87 (1991), Remscheid et al. (Applied and Environmental Microbiology 60,126-132 (1994), in LaBarre et al., (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application WO 96/15246, in Malumbres et al., (Gene 134, 15-24 (1993)), Japanese Laid-Open Patent Publication JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 1991-195 (1998)), Makrides (Microbiological Reviews 60: 512-538 (1996) and in well-known textbooks of genetics and molecular biology.
Zur Herstellung geeigneter Produktionsstämme werden Expressionskonstrukte oder Vektoren verwendet, enthaltend unter der genetischen Kontrolle regulativer Nu- kleinsäuresequenzen eine für eine gewünschte Enzymaktivität kodierende Nukleinsäu- resequenz. Vorzugsweise umfassen solche Konstrukte 5'-stromaufwärts von der jeweiligen kodierenden Sequenz einen Promotor und 3'-stromabwärts eine Terminatorsequenz sowie gegebenenfalls weitere übliche regulative Elemente, und zwar jeweils operativ verknüpft mit der kodierenden Sequenz. Unter einer „operativen Verknüpfung" versteht man die sequentielle Anordnung von Promotor, kodierender Sequenz, Terminator und gegebenenfalls weiterer regulativer Elemente derart, dass jedes der regulativen Elemente seine Funktion bei der Expression der kodierenden Sequenz bestimmungsgemäß erfüllen kann. Beispiele für operativ verknüpfbare Sequenzen sind Aktivierungssequenzen sowie Enhancer und dergleichen. Weitere regulative Elemente umfassen selektierbare Marker, Amplifikationssignale, Replikationsursprünge und dergleichen. Geeignete regulatorische Sequenzen sind z.B. beschrieben in Goeddel, Ge- ne Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).For the production of suitable production strains, expression constructs or vectors are used which contain, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a desired enzyme activity. Preferably, such constructs comprise a promoter 5'-upstream of the respective coding sequence and a terminator sequence 3'-downstream, and optionally other common regulatory elements, each operably linked to the coding sequence. "Operational linkage" is understood to mean the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended Enhancers and the like Further regulatory elements include selectable markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in Goeddel, GE. Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Zusätzlich zu den artifiziellen Regulationssequenzen kann die natürliche Regulations- sequenz vor dem eigentlichen Strukturgen noch vorhanden sein. Durch genetische Veränderung kann diese natürliche Regulation gegebenenfalls ausgeschaltet und die Expression der Gene erhöht oder erniedrigt werden. Das Genkonstrukt kann aber auch einfacher aufgebaut sein, das heißt es werden keine zusätzlichen Regulationssignale vor das Strukturgen insertiert und der natürliche Promotor mit seiner Regulation wird nicht entfernt. Statt dessen wird die natürliche Regulationssequenz so mutiert, dass keine Regulation mehr erfolgt und die Genexpression gesteigert oder verringert wird. Die Nukleinsäuresequenzen können in einer oder mehreren Kopien im Genkonstrukt enthalten sein.In addition to the artificial regulatory sequences, the natural regulatory sequence may still be present before the actual structural gene. By genetic modification, this natural regulation can optionally be switched off and the expression of the genes increased or decreased. However, the gene construct can also be constructed more simply, that is, no additional regulatory signals are inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation stops and gene expression is increased or decreased. The nucleic acid sequences may be contained in one or more copies in the gene construct.
Beispiele für brauchbare Promotoren sind: die Promotoren, ddh, amy, lysC, dapA, lysA aus Corynebacterium glutamicum, aber auch gram-positiven Promotoren SPO2 wie sie in Bacillus Subtilis and Its Closest Relatives, Sonenshein, Abraham L., Hoch, James A., Losick, Richard; ASM Press, District of Columbia, Washington und Patek M. Eik- manns BJ. Patek J. Sahm H. Microbiology. 142 1297-309, 1996 beschrieben sind, oder aber auch cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ipp-lac-, laclq-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, λ-PR- oder im λ-PL-Promotor, die vorteilhafterweise in gram-negativen Bakterien Anwendung finden. Bevorzugt ist auch die Verwendung induzierbarer Promotoren, wie z.B. licht- und insbesondere temperaturinduzierbarer Promotoren, wie der PrPr Promotor. Prinzipiell können alle natürlichen Promotoren mit ihren Regulationssequen- zen verwendet werden. Darüber hinaus können auch synthetische Promotoren, wie Mehrfachpromotoren (vgl. z.B. WO2006/069711 ) vorteilhaft verwendet werden.Examples of useful promoters are: the promoters, ddh, amy, lysC, dapA, lysA from Corynebacterium glutamicum, but also gram-positive promoters SPO2 as described in Bacillus Subtilis and Its Closest Relatives, Sonenshein, Abraham L., Hoch, James A. , Losick, Richard; ASM Press, District of Columbia, Washington and Patek M. Eikmanns BJ. Patek J. Sahm H. Microbiology. 142 1297-309, 1996, or else cos, tac, trp, tet, trp tet, lpp, lac, lpp-lac, laclq, T7, T5, T3 , gal, trc, ara, SP6, λ-PR or in the λ PL promoter, which are advantageously used in gram-negative bacteria. Also preferred is the use of inducible promoters, such as light and in particular temperature-inducible promoters, such as the P r P r promoter. In principle, all natural promoters with their regulatory sequences can be used. In addition, synthetic promoters, such as multiple promoters (cf., for example, WO2006 / 069711) can also be used to advantage.
Die genannten regulatorischen Sequenzen sollen die gezielte Expression der Nukleinsäuresequenzen ermöglichen. Dies kann beispielsweise je nach Wirtsorganismus be- deuten, dass das Gen erst nach Induktion exprimiert oder überexprimiert wird, oder dass es sofort exprimiert und/oder überexprimiert wird.The said regulatory sequences are intended to enable targeted expression of the nucleic acid sequences. Depending on the host organism, this may mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
Die regulatorischen Sequenzen bzw. Faktoren können dabei vorzugsweise die Expression positiv beeinflussen und dadurch erhöhen oder erniedrigen. So kann eine Verstär- kung der regulatorischen Elemente vorteilhafterweise auf der Transkriptionsebene erfolgen, indem starke Transkriptionssignale wie Promotoren und/oder "Enhancer" ver- wendet werden. Daneben ist aber auch eine Verstärkung der Translation möglich, indem beispielsweise die Stabilität der mRNA verbessert wird.The regulatory sequences or factors can thereby preferably positively influence the expression and thereby increase or decrease. Thus, an enhancement of the regulatory elements can advantageously take place at the level of transcription, by using strong transcription signals such as promoters and / or enhancers. be used. In addition, however, an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
Die Herstellung einer Expressionskassette erfolgt durch Fusion eines geeigneten Pro- motors, einer geeigneten Shine-Dalgarnow-Sequenz mit einer Lysinbiosynthese- Nukleotidsequenz sowie einem geeigneten Terminationssignal. Dazu verwendet man gängige Rekombinations- und Klonierungstechniken, wie sie beispielsweise in Current Protocols in Molecular Biology, 1993, John Wiley & Sons, Incorporated, New York New York, PCR Methods, Gelfand, David H., Innis, Michael A., Sninsky, John J. 1999, Aca- demic Press, Incorporated, California, San Diego, ., PCR Cloning Protocols, Methods in Molecular Biology Ser., Vol. 192, 2nd ed., Humana Press, New Jersey, Totowa. T. Maniatis, E. F. Fritsch und J. Sambrook, Molecular Cloning: A Laboratory Manual, CoId Spring Harbor Laboratory, CoId Spring Harbor, NY (1989) sowie in TJ. Silhavy, M. L. Berman und L.W. Enquist, Experiments with Gene Fusions, CoId Spring Harbor Labo- ratory, CoId Spring Harbor, NY (1984) und in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987) beschrieben sind.An expression cassette is produced by fusion of a suitable promoter, a suitable Shine-Dalgarnow sequence with a lysine biosynthesis nucleotide sequence and a suitable termination signal. Common recombinant and cloning techniques are used, as described, for example, in Current Protocols in Molecular Biology, 1993, John Wiley & Sons, Incorporated, New York New York, PCR Methods, Gelfand, David H., Innis, Michael A., Sninsky, John J. 1999, Antibacterial Press, Incorporated, California, San Diego,, PCR Cloning Protocols, Methods in Molecular Biology Ser., Vol. 192, 2nd ed., Humana Press, New Jersey, Totowa. T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Colard Spring Harbor Laboratory, ColD Spring Harbor, NY (1989) and TJ. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusion, CoId Spring Harbor Laboratory, ColD Spring Harbor, NY (1984), and Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
Das rekombinante Nukleinsäurekonstrukt bzw. Genkonstrukt wird zur Expression in einem geeigneten Wirtsorganismus vorteilhafterweise in einen wirtsspezifischen Vektor insertiert, der eine optimale Expression der Gene im Wirt ermöglicht. Vektoren sind dem Fachmann wohl bekannt und können beispielsweise aus "Cloning Vectors" (Pou- wels P. H. et al., Hrsg, Elsevier, Amsterdam-New York-Oxford, 1985) entnommen werden. Unter Vektoren sind außer Plasmiden auch alle anderen dem Fachmann bekann- ten Vektoren, wie beispielsweise Phagen, Transposons, IS-Elemente, Phasmide, Cosmide, und lineare oder zirkuläre DNA zu verstehen. Diese Vektoren können autonom im Wirtsorganismus repliziert oder chromosomal repliziert werden.The recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector for expression in a suitable host organism, which enables optimal expression of the genes in the host. Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985). In addition to plasmids, vectors include all other vectors known to the person skilled in the art, such as, for example, phages, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally.
Als Plasmide eignen sich solche, die in coryneformen Bakterien repliziert werden. Zahl- reiche bekannte Plasmidvektoren, wie z. B. pZ1 (Menkel et al., Applied and Environ- mental Microbiology (1989) 64: 549-554), pEKExi (Eikmanns et al., Gene 102: 93-98 (1991 )) oder pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991 )) beruhen auf den kryptischen Plasmiden pHM1519, pBL1 oder pGA1. Andere Plasmidvektoren, wie z. B. pCLiK5MCS, oder solche, die auf pCG4 (US-A 4,489,160) oder pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) oder pAG1 (US-A 5,158,891 ) beruhen, können in gleicher Weise verwendet werden. Weiterhin eignen sich auch solche Plasmidvektoren mit Hilfe derer man das Verfahren der Genamplifikation durch Integration in das Chromosom anwenden kann, so wie es beispielsweise von Remscheid et al. (Applied and Environmental Microbiology 60,126- 132 (1994)) zur Duplikation bzw. Amplifikation des hom-thrB-Operons beschrieben wurde. Bei dieser Methode wird das vollständige Gen in einen Plasmidvektor kloniert, der in einem Wirt (typischerweise E. coli), nicht aber in C. glutamicum replizieren kann. Als Vektoren kommen beispielsweise pSUP301 (Simon et al., Bio/ Technology 1 ,784- 791 (1983)), pK18mob oder pK19mob (Schäfer et al., Gene 145,69-73 (1994)), Ber- nard et al., Journal ofMolecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al. 1991 , Journal of Bacteriology 173: 4510-4516) oder pBGSδ (Spratt et al.,1986, Gene 41 : 337-342) in Frage. Der Plasmidvektor, der das zu amplifizierende Gen enthält, wird anschließend durch Transformation in den gewünschten Stamm von C. glutamicum überführt. Methoden zur Transformation sind beispielsweise bei Thierbach et al. (Ap- plied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican und Shivnan (Bio- technology 7, 1067-1070 (1989)) und Tauch et al. (FEMS Microbiological Letters 123,343-347 (1994)) beschrieben.Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such. PZKE (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExi (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al , Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such as. B. pCLiK5MCS, or those based on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891), can be in be used in the same way. Furthermore, those plasmid vectors by means of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum. Examples of vectors which are used are pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), Bernard et al. , Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173: 4510-4516) or pBGSδ (Spratt et al., 1986, Gene 41: 337-342) Question. The plasmid vector containing the gene to be amplified is then transformed into the desired strain of C. glutamicum by transformation. Methods for transformation are described by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Biotechnology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123,343-347 (1994)).
Enzyme können durch Mutationen in den korrespondierenden Genen derart in ihrer Aktivität beeinflusst werden, dass es zu einer teilweisen oder vollständigen Verringerung der Reaktionsgeschwindigkeit der enzymatischen Reaktion kommt. Beispiele für solche Mutationen sind dem Fachmann bekannt (Motoyama H. Yano H. Terasaki Y. Anazawa H. Applied & Environmental Microbiology. 67:3064-70, 2001 , Eikmanns BJ. Eggeling L. Sahm H. Antonie van Leeuwenhoek. 64:145-63, 1993-94.). Mit dieser Maßnahme können z.B. mit der erfindungsgemäßen Lysinbiosynthese konkurrierende Reaktionen ausgeschaltet oder verlangsamt werden. (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krum- phanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).Enzymes can be affected in their activity by mutations in the corresponding genes such that there is a partial or complete reduction in the reaction rate of the enzymatic reaction. Examples of such mutations are known to those skilled in the art (Motoyama H. Yano H. Terasaki Y. Anazawa H. Applied & Environmental Microbiology 67: 3064-70, 2001, Eikmanns BJ, Eggeling L. Sahm H. Antonie van Leeuwenhoek 64: 145 -63, 1993-94.). With this measure, e.g. With the lysine biosynthesis according to the invention competing reactions are eliminated or slowed down. (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
Zusätzlich kann es für die Produktion von L-Lysin vorteilhaft sein, neben einer Expression bzw. Verstärkung der Lysinbiosynthese-Gene eines oder mehrere Enzyme vorgeschalteter Biosyntheseweges, wie z.B. des Pentose-Phosphat-Stoffwechsels, des Zitronensäure-Zyklus, oder des Aminosäure-Exports zu verstärken.In addition, for the production of L-lysine, it may be advantageous, in addition to expression or amplification of the lysine biosynthesis genes, to have one or more enzymes of an upstream biosynthetic pathway, such as e.g. pentose-phosphate metabolism, the citric acid cycle, or amino acid export.
Die erfindungsgemäß verwendeten Mikroorganismen können kontinuierlich oder diskontinuierlich im batch- Verfahren (Satzkultivierung) oder im fed batch (Zulaufverfah- ren) oder repeated fed batch Verfahren (repetitives Zulaufverfahren) zur Produktion von L-Lysin, kultiviert werden. Eine Zusammenfassung über bekannte Kultivierungsmethoden ist im Lehrbuch von Chmiel (Bioprozeßtechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991 )) oder im Lehrbuch von Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)) zu finden.The microorganisms used according to the invention can be used continuously or discontinuously in the batch process (batch cultivation) or in the fed batch (feed process). Ren) or repeated fed batch process (repetitive feed method) for the production of L-lysine, be cultured. A summary of known cultivation methods is in the textbook by Chmiel (Bioprozesstechnik 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) Find.
Das zu verwendende Kulturmedium hat in geeigneter Weise den Ansprüchen der jeweiligen Stämme zu genügen. Beschreibungen von Kulturmedien verschiedener Mik- roorganismen sind im Handbuch "Manual of Methods für General Bacteriology" der American Society für Bacteriology (Washington D. C, USA, 1981 ) enthalten.The culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society of Bacteriology (Washington D.C, USA, 1981).
Diese erfindungsgemäß einsetzbaren Medien umfassen gewöhnlich eine oder mehreren Kohlenstoffquellen, Stickstoffquellen, anorganische Salze, Vitamine und/oder Spu- renelemente.These media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
Bevorzugte Kohlenstoffquellen sind Zucker, wie Mono-, Di- oder Polysaccharide. Sehr gute Kohlenstoffquellen sind beispielsweise Glucose, Fructose, Mannose, Galactose, Ribose, Sorbose, Ribulose, Lactose, Maltose, Saccharose, Raffinose, Stärke oder CeI- lulose. Man kann Zucker auch über komplexe Verbindungen, wie Melassen, oder andere Nebenprodukte der Zucker-Raffinierung zu den Medien geben. Es kann auch vorteilhaft sein, Gemische verschiedener Kohlenstoffquellen zuzugeben. Andere mögliche Kohlenstoffquellen sind Öle und Fette wie z. B. Sojaöl, Sonnenblumenöl, Erdnußöl und Kokosfett, Fettsäuren wie z. B. Palmitinsäure, Stearinsäure oder Linolsäure, Alkohole wie z. B. Glycerin, Methanol oder Ethanol und organische Säuren wie z. B. Essigsäure oder Milchsäure.Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add mixtures of different carbon sources. Other possible sources of carbon are oils and fats such. As soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such. As palmitic acid, stearic acid or linoleic acid, alcohols such. As glycerol, methanol or ethanol and organic acids such. As acetic acid or lactic acid.
Stickstoffquellen sind gewöhnlich organische oder anorganische Stickstoffverbindungen oder Materialien, die diese Verbindungen enthalten. Beispielhafte Stickstoffquellen umfassen Ammoniak-Gas bzw. Ammoniakwasser oder Ammoniumsalze, wie Ammoniumsulfat, Ammoniumchlorid, Ammoniumphosphat, Ammoniumcarbonat, Ammonium- carbamat oder Ammoniumnitrat, Nitrate, Harnstoff, Aminosäuren oder komplexe Stickstoffquellen, wie Maisquellwasser, Sojamehl, Sojaprotein, Hefeextrakt, Fleischextrakt und andere. Die Stickstoffquellen können einzeln oder als Mischung verwendet wer- den. Anorganische Salzverbindungen, die in den Medien enthalten sein können, umfassen die Chlorid-, Phosphat-, Carbonat- oder Sulfatsalze von Calcium, Magnesium, Natrium, Kobalt, Molybdän, Kalium, Mangan, Zink, Kupfer und Eisen sowie Borsäure.Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds. Exemplary nitrogen sources include ammonia gas or ammonia water or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium carbamate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture. Inorganic salt compounds that may be included in the media include the chloride, phosphate, carbonate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron, and boric acid.
Als Schwefelquelle können anorganische schwefelhaltige Verbindungen wie beispielsweise Sulfate, Sulfite, Dithionite, Tetrathionate, Thiosulfate, Sulfide aber auch organische Schwefelverbindungen, wie Mercaptane und Thiole, verwendet werden.As sulfur source inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic sulfur compounds, such as mercaptans and thiols can be used.
Als Phosphorquelle können Phosphorsäure, Kaliumdihydrogenphosphat oder Dikali- umhydrogenphosphat oder die entsprechenden Natrium-haltigen Salze verwendet werden.Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
Chelatbildner können zum Medium gegeben werden, um die Metallionen in Lösung zu halten. Besonders geeignete Chelatbildner umfassen Dihydroxyphenole, wie Catechol oder Protocatechuat, oder organische Säuren, wie Citronensäure.Chelating agents can be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
Die erfindungsgemäß eingesetzten Kulturmedien enthalten üblicherweise auch andere Wachstumsfaktoren, wie Vitamine oder Wachstumsförderer, zu denen beispielsweise Biotin, Riboflavin, Thiamin, Folsäure, Nikotinsäure, Panthothenat und Pyridoxin gehö- ren. Wachstumsfaktoren und Salze stammen häufig von komplexen Medienkomponenten, wie Hefeextrakt, Melassen, Maisquellwasser und dergleichen. Dem Kulturmedium können überdies geeignete Vorstufen zugesetzt werden. Die genaue Zusammensetzung der Medienverbindungen hängt stark vom jeweiligen Experiment ab und wird für jeden spezifischen Fall individuell entschieden. Information über die Medienoptimie- rung ist erhältlich aus dem Lehrbuch "Applied Microbiol. Physiology, A Practical Ap- proach" (Hrsg. P.M. Rhodes, P. F. Stanbury, IRL Press (1997) S. 53-73, ISBN 0 19 963577 3). Wachstumsmedien lassen sich auch von kommerziellen Anbietern beziehen, wie Standard 1 (Merck) oder BHI (Brain heart infusion, DIFCO) und dergleichen.The culture media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often originate from complex media components, such as yeast extract, molasses, corn steep liquor and the same. In addition, suitable precursors can be added to the culture medium. The exact composition of the media compounds will depend heavily on the particular experiment and will be decided on a case by case basis. Information about the media optimization is available from the textbook "Applied Microbiol. Physiology, A Practical Apache" (ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media may also be obtained from commercial suppliers such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
Sämtliche Medienkomponenten werden, entweder durch Hitze (z.B. 20 min bei 1 bar Überdruck (2 bar absolut) und 1210C) oder durch Sterilfiltration, sterilisiert. Die Komponenten können entweder zusammen oder nötigenfalls getrennt sterilisiert werden. Sämtliche Medienkomponenten können zu Beginn der Anzucht zugegen sein oder wahlfrei kontinuierlich oder portionsweise hinzugegeben werden. Die Temperatur der Kultur liegt normalerweise zwischen 15°C und 45°C, vorzugsweise bei 25°C bis 400C und kann während des Experimentes konstant gehalten oder verändert werden. Der pH-Wert des Mediums sollte im Bereich von 5 bis 8,5, vorzugsweise um 7,0 liegen. Der pH-Wert für die Fermentation lässt sich während der Fermentation durch Zugabe von basischen Verbindungen wie z.B. Natriumhydroxid, Kaliumhydroxid, Natriumhydrogencarbonat, Ammoniak bzw. Ammoniakwasser oder saure Verbindungen wie Phosphorsäure, Salzsäure oder Schwefelsäure kontrollieren. Zur Kontrolle der Schaumentwicklung können Antischaummittel wie z. B. Fettsäurepolyglykolester, PoIy- alkylenglykole, Silikone oder andere eingesetzt werden (siehe z.B. Biotechnol. Progr. 2007, 23, 767-784). Zur Aufrechterhaltung der Stabilität von Plasmiden können dem Medium geeignete selektiv wirkende Stoffe, wie z. B. Antibiotika, hinzugefügt werden. Um aerobe Bedingungen aufrechtzuerhalten, werden Sauerstoff oder Sauerstoffhaltige Gasmischungen, wie z. B. Umgebungsluft, in die Kultur eingetragen. Die Temperatur der Kultur liegt normalerweise bei 200C bis 45°C. Die Kultur wird solange fort- gesetzt, bis sich ein Maximum des gewünschten Produktes gebildet hat. Dieses Ziel wird normalerweise innerhalb von 10 Stunden bis 160 Stunden erreicht.All media components are sterilized either by heat (eg 20 min at 1 bar overpressure (2 bar absolute) and 121 ° C.) or by sterile filtration. The components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or optionally added continuously or in portions. The temperature of the culture is usually between 15 ° C and 45 ° C, preferably at 25 ° C to 40 0 C and can be kept constant or changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH for the fermentation can be controlled during the fermentation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, ammonia or ammonia water or acidic compounds such as phosphoric acid, hydrochloric acid or sulfuric acid. To control the foam development antifoams such. As fatty acid polyglycol esters, polyalkylene glycols, silicones or others are used (see, for example Biotechnol. Progr., 2007, 23, 767-784). To maintain the stability of plasmids, the medium can be selected selectively acting substances such. As antibiotics, are added. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such. B. ambient air, registered in the culture. The temperature of the culture is usually from 20 0 C to 45 ° C. The culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.
Die so erhaltenen, insbesondere L-Lysin oder DAP enthaltenden, Fermentationsbrühen haben üblicherweise eine Trockenmasse von 3 bis 20 Gew.-%.The fermentation broths thus obtained, in particular containing L-lysine or DAP, usually have a dry matter content of from 3 to 20% by weight.
Vorteilhaft ist außerdem auch, wenn die Fermentation zumindest am Ende, insbesondere jedoch über mindestens 30% der Fermentationsdauer zuckerlimitiert gefahren wird. Das heißt, dass während dieser Zeit die Konzentration an verwertbarem Zucker im Fermentationsmedium auf > 0 bis 3 g/l gehalten, beziehungsweise abgesenkt wird.It is also advantageous if the fermentation is driven sugar-limited at least at the end, but especially over at least 30% of the fermentation period. This means that during this time the concentration of utilizable sugar in the fermentation medium is kept at> 0 to 3 g / l, or lowered.
Die Fermentationsbrühe wird anschließend weiterverarbeitet. Je nach Anforderung kann die Biomasse ganz oder teilweise durch Separationsmethoden, wie z. B. Zentri- fugation, Filtration, Dekantieren, Flockung oder eine Kombination dieser Methoden aus der Fermentationsbrühe entfernt oder vollständig in ihr belassen werden. Bevorzugt ist eine Abtrennung der Biomasse.The fermentation broth is then further processed. Depending on the requirement, the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation, flocculation or a combination of these methods are removed from the fermentation broth or completely left in her. A separation of the biomass is preferred.
2.3 Aufarbeitung der DAP-haltigen Fermentationsbrühe2.3 Work-up of the DAP-containing fermentation broth
Anschließend kann die Fermentationsbrühe mit bekannten Methoden, wie z. B. mit Hilfe eines Rotationsverdampfers, Dünnschichtverdampfers, Fallfilmverdampfers, durch Umkehrosmose, oder durch Nanofiltration, eingedickt beziehungsweise aufkon- zentriert werden. Sofern erforderlich, können eventuell durch die Aufkonzentrierung ausgefallene Salze beispielsweise durch Filtration oder Zentrifugation abgetrennt werden. Diese aufkonzentrierte Fermentationsbrühe kann anschließend in der erfindungsgemäßen Weise aufgearbeitet werden, um DAP zu erhalten. Für die Aufarbeitung im Sinne der vorliegenden Erfindung ist eine solche Aufkonzentrierung möglich, aber nicht zwingend erforderlich.Subsequently, the fermentation broth with known methods, such as. B. with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or aufkon- be centered. If necessary, any salts precipitated by the concentration may be separated by, for example, filtration or centrifugation. This concentrated fermentation broth can then be worked up in the manner according to the invention to obtain DAP. For the purposes of the present invention, such a concentration is possible, but not absolutely necessary.
Erfindungsgemäß wird DAP mit Hilfe eines organischen Extraktionsmittels aus der Fermentationsbrühe extrahiert. Dabei verwendet man insbesondere ein organisches Lösungsmittel mit Mischungslücke mit Wasser, das möglichst polar und im Alkalischen stabil ist, wie insbesondere ein polares, insbesondere dipolar-protisches, organisches Lösungsmittel. Geeignete Lösungsmittel sind insbesondere zyklische oder offenketti- ge, gegebenenfalls verzweigte Alkanole mit 3 bis 8 C-Atomen, wie insbesondere, n- und iso-Propanol, n-, sec- und iso-Butanol, oder Cyclohexanol, sowie n-Pentanol, n- Hexanol- n-Heptanol, n-Oktanol, 2-Oktanol und die ein- oder mehrfach verzweigten isomeren Formen davon. n-Butanol ist dabei besonders zu erwähnen.According to the invention, DAP is extracted from the fermentation broth using an organic extractant. In particular, an organic solvent having a miscibility gap with water, which is as polar as possible and stable in the alkaline, is used, in particular a polar, in particular dipolar protic, organic solvent. Suitable solvents are, in particular, cyclic or open-chain, optionally branched alkanols having 3 to 8 C atoms, such as, in particular, n- and iso-propanol, n-, sec- and isobutanol, or cyclohexanol, and also n-pentanol, n - Hexanol n-heptanol, n-octanol, 2-octanol and the one or more branched isomeric forms thereof. N-butanol is especially worth mentioning.
In einer bevorzugten Ausführungsform wird die Extraktion und/oder die anschließende Phasentrennung diskontinuierlich bei erhöhter Temperatur durchgeführt, wobei die Temperatur durch die Siedepunkte von Wasser und des Extraktionsmittels bzw. sich möglicherweise bildender Azeotrope begrenzt ist. Mit n-Butanol als Extraktionsmittel könnten Extraktion und Phasentrennung z.B. bei etwa 25-90 0C oder bevorzugt bei 40-70 0C durchgeführt werden. Für die Extraktion werden die beiden Phasen gerührt, bis sich das Verteilungsgleichgewicht eingestellt hat, z.B. über einen Zeitraum von 10 Sekunden bis 2 Stunden, bevorzugt 5 bis 15Min . Anschließend lässt man die Phasen absitzen, bis sich die Phasen vollständig getrennt haben; dies erfolgt vorzugsweise über einen Zeitraum von 10 Sekunden bis 5 Stunden, wie z.B. 15 bis 120 oder 30 bis 90 Minuten, insbesondere auch bei einer Temperatur im Bereich von etwa 25-90 0C oder 40-70 0C im Fall von n-Butanol.In a preferred embodiment, the extraction and / or the subsequent phase separation is carried out batchwise at elevated temperature, the temperature being limited by the boiling points of water and of the extractant or possibly forming azeotropes. With n-butanol as the extraction agent extraction and phase separation, for example, at about 25-90 0 C or preferably at 40-70 0 C could be performed. For the extraction, the two phases are stirred until the distribution equilibrium has settled, for example over a period of 10 seconds to 2 hours, preferably 5 to 15 minutes. Subsequently, the phases are allowed to settle until the phases have completely separated; This is preferably carried out over a period of 10 seconds to 5 hours, such as 15 to 120 or 30 to 90 minutes, especially at a temperature in the range of about 25-90 0 C or 40-70 0 C in the case of n-butanol ,
In weiteren bevorzugten Ausführungsformen wird die Extraktion des DAPs aus der Fermentationsbrühe kontinuierlich mehrstufig (z.B. in Mixer-Settler-Kombinationen) oder kontinuierlich in einer Extraktionskolonne durchgeführt.In further preferred embodiments, the extraction of the DAP from the fermentation broth is carried out continuously in multiple stages (e.g., in mixer-settler combinations) or continuously in an extraction column.
Die apparative Ausgestaltung der erfindungsgemäß einsetzbaren Extraktionskolonnen können vom Fachmann für die jeweils zu trennenden Phasen im Rahmen routinemä- ßigen Optimierungsarbeiten festgelegt werden. Geeignet sind grundsätzlich Extraktionskolonnen ohne Leistungseintrag oder Extraktionskolonnen mit Leistungseintrag, wie z. B. pulsierte Kolonnen oder Kolonnen mit rotierenden Einbauten. Der Fachmann kann auch im Rahmen routinemäßiger Arbeiten Art und Materialen von Einbauten, wie Siebböden, und Kolonnen-Füllkörper, zur Optimierung der Phasentrennung in geeigneter Weise auswählen. Die theoretischen Grundlagen der Flüssig-Flüssig-Extraktion kleiner Moleküle sind allgemein bekannt (vgl. z.B. H. -J. Rehm and G. Reed, Eds., (1993), Biotechology, Volume 3 Bioprocessing, Chapter 21 , VCH, Weinheim). Die Ausgestaltung von industriell anwendbaren Extraktionskolonnen ist beispielsweise be- schrieben in Lo et al., Eds., (1983) Handbook of Solvent Extraction, JohnWiley& Sons, New York. Auf die Offenbarung obiger Lehrbücher wird ausdrücklich Bezug genommen.The apparatus configuration of the extraction columns which can be used according to the invention can be determined by the person skilled in the art for the phases to be separated in each case in a routine manner. constant optimization work. Suitable are in principle extraction columns without power input or extraction columns with power input, such. B. pulsed columns or columns with rotating internals. The person skilled in the art can also select the type and materials of internals, such as sieve trays, and column packings in the course of routine work, in order to optimize the phase separation in a suitable manner. The theoretical foundations of liquid-liquid extraction of small molecules are well known (see, for example, H.-J. Rehm and G. Reed, Eds., (1993), Biotechology, Volume 3 Bioprocessing, Chapter 21, VCH, Weinheim). The design of industrially applicable extraction columns is described, for example, in Lo et al., Eds., (1983) Handbook of Solvent Extraction, John Wiley & Sons, New York. The disclosure of the above textbooks is expressly incorporated by reference.
Nach der Phasentrennung erfolgt in an sich bekannter Weise die Isolierung und Auf- reinigung des DAPs aus der DAP-haltigen Extraktphase. Mögliche Maßnahmen zur DAP-Gewinnung sind, ohne darauf beschränkt zu sein, insbesondere die Destillation, die Fällung als Salz mit geeigneten organischen oder anorganischen Säuren, oder Kombinationen solcher geeigneter Maßnahmen.After the phase separation, the isolation and purification of the DAP from the DAP-containing extract phase are carried out in a manner known per se. Possible measures for DAP recovery include, but are not limited to, distillation, precipitation as a salt with suitable organic or inorganic acids, or combinations of such suitable measures.
Die Destillation kann dabei kontinuierlich oder satzweise (diskontinuierlich) erfolgen. Es können eine einzelne oder mehrere miteinander gekoppelte Destillationskolonnen verwendet werden. Die apparative Ausgestaltung der Destillationskolonne und die Festlegung der Betriebsparameter obliegt dem Fachmann. Die jeweils eingesetzten Destillationskolonnen können dabei in an sich bekannter Weisen realisiert werden (siehe z. B. Sattler, Thermische Trennverfahren, 2. Auflage 1995, Weinheim, S. 135ff; Perry's Chemical Engineers Handbook, 7. Auflage 1997, New York, Section 13). So können die eingesetzten Destillationskolonnen z.B. trennwirksame Einbauten enthalten, wie Trennböden, z. B. Lochböden, Glockenböden oder Ventilböden, geordnete Packungen, z. B. Blech- oder Gewebepackungen, oder regellose Schüttungen von Füllkörpern. Die in der/den eingesetzten Kolonne(n) notwendige Stufenzahl und das Rücklaufverhältnis richten sich im Wesentlichen nach den Reinheitsanforderungen und der relativen Siedelage der zu trennenden Flüssigkeiten, wobei der Fachmann die konkreten Ausle- gungs- und Betriebsdaten nach bekannten Methoden ermitteln kann.The distillation can be carried out continuously or batchwise (batchwise). A single or more coupled distillation columns can be used. The apparatus design of the distillation column and the determination of the operating parameters is the expert. The distillation columns used in each case can be realized in a manner known per se (see, for example, Sattler, Thermal Separation Methods, 2nd Edition 1995, Weinheim, page 135 et seq., Perry's Chemical Engineers Handbook, 7th Edition 1997, New York, Section 13 ). Thus, the distillation columns used may be e.g. separating internals included, such as separating trays, z. As perforated plates, bubble trays or valve trays, ordered packs, z. As sheet or tissue packs, or random beds of packing. The number of stages and the reflux ratio necessary in the column (s) used depend essentially on the purity requirements and the relative boiling position of the liquids to be separated, with the person skilled in the art being able to determine the specific design and operating data by known methods.
Die Fällung als Salz kann durch Zugabe geeigneter organischer oder anorganischer Säuren herbeigeführt werden, wie zum Beispiel Schwefelsäure, Salzsäure, Phosphor- säure, Essigsäure, Ameisensäure, Kohlensäure, Oxalsäure, usw. In einer weiteren bevorzugten Ausführungsform wird eine organische Dicarbonsäure verwendet, die zur Bildung eines Salzes führt, welches direkt oder nach Aufreinigung, z.B. durch Umkristallisation, in einer nachfolgenden Polykondensation zum Polyamid eingesetzt werden kann. Solche Dicarbonsäuren können insbesondere C4-Ci2-Dicarbonsäuren sein.The precipitation as a salt can be brought about by addition of suitable organic or inorganic acids, such as, for example, sulfuric acid, hydrochloric acid, phosphoric acid. acid, acetic acid, formic acid, carbonic acid, oxalic acid, etc. In a further preferred embodiment, an organic dicarboxylic acid is used which leads to the formation of a salt which can be used directly or after purification, for example by recrystallization, in a subsequent polycondensation to the polyamide. Such dicarboxylic acids may be C 4 -C 2 dicarboxylic acids in particular.
Die bei der Extraktion angefallene organische DAP-Phase kann auch chromatographisch aufgearbeitet werden. Für die Chromatographie wird die DAP-Phase auf ein geeignetes Harz aufgetragen, z.B. einen stark oder schwach sauren lonentauscher, (etwa Lewatit 1468 S, Dowex Marathon C, Amberlyst 1 19 Wet oder andere), wobei das gewünschte Produkt oder die Verunreinigungen ganz oder teilweise auf dem Chromatographieharz zurückgehalten werden. Diese Chromatographieschritte können nötigenfalls wiederholt werden, wobei die gleichen oder andere Chromatographieharze ver- wendet werden. Der Fachmann ist in der Auswahl der geeigneten Chromatographieharze und ihrer wirksamsten Anwendung bewandert. Das gereinigte Produkt kann durch Filtration oder Ultrafiltration konzentriert und bei geeigneter Temperatur aufbewahrt werden.The organic DAP phase obtained during the extraction can also be worked up by chromatography. For chromatography, the DAP phase is applied to a suitable resin, e.g. a strong or weakly acidic ion exchanger (such as Lewatit 1468 S, Dowex Marathon C, Amberlyst 1 19 Wet or others) with the desired product or impurities retained in whole or in part on the chromatography resin. If necessary, these chromatographic steps can be repeated using the same or different chromatography resins. The person skilled in the art is familiar with the choice of suitable chromatography resins and their most effective use. The purified product may be concentrated by filtration or ultrafiltration and stored at a suitable temperature.
Die Identität und Reinheit der isolierten Verbindung(en) kann durch Techniken des Standes der Technik bestimmt werden. Diese umfassen Hochleistungs- Flüssigkeitschromatographie (HPLC), Gaschromatographie (GC), spektroskopische Verfahren, Färbeverfahren, Dünnschichtchromatographie, NIRS, Enzymtest oder mikrobiologische Tests. Diese Analyseverfahren sind zusammengefasst in: Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 1 1 27-32; und Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ullmann's Ency- clopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S. 89-90, S. 521- 540, S. 540-547, S. 559-566, 575-581 und S. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.The identity and purity of the isolated compound (s) can be determined by techniques of the prior art. These include high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopic methods, staining procedures, thin layer chromatography, NIRS, enzyme assay or microbiological assays. These analytical methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 1 1 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, pp. 575-581 and pp. 581- 587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.
Die Erfindung wird nun anhand der folgenden nicht-limitierenden Beispiele und unter Bezugnahme auf beiliegende Figuren näher beschrieben. EXPERIMENTELLER TEILThe invention will now be described in more detail by way of the following non-limiting examples and with reference to accompanying figures. EXPERIMENTAL PART
Fermentationsbeispiel 1 :Fermentation Example 1:
Aus einer Stammkultur eines an sich bekannten DAP-Produktionsstammes (vgl. WO 2007/113127, Beispiel 2, Seite 14; worauf hiermit ausdrücklich Bezug genommen wird) (Lagerung bei -800C) wurde eine Teilmenge Zellen entnommen, auf Festmedium (jeweilige Medienzusammensetzungen vgl. Tabelle 2) in einer Petrischale (Kultur 1 ) ausgestrichen und anschließend bei 300C für 72 h inkubiert. Die so gewonnenen Zellen wurden in 0,9 %-iger NaCI-Lösung aufgenommen, erneut auf Festmedium in einer Petrischale (Kultur 2) ausgestrichen und anschließend bei 30°C für weitere 24 h inkubiert. Anschließend wurden die Zellen mit einer Impföse von der Petrischale (Kultur 2) in 200 mL eines 2L-Schüttelkolben mit 2 Schikanen (Zusammensetzung analog Platten- und Batchmedium) überimpft und bei 300C auf einem Orbitalschüttler bei einer Dreh- zahl von 250 Upm für 24 h inkubiert.From a stock culture of a known DAP production strain (see WO 2007/113127, Example 2, page 14, which is hereby incorporated by reference) (storage at -80 0 C), a subset of cells was removed, on solid medium (respective media compositions see Table 2) in a Petri dish (culture 1) and then incubated at 30 0 C for 72 h. The cells thus obtained were taken up in 0.9% NaCl solution, streaked again on solid medium in a Petri dish (culture 2) and then incubated at 30 ° C. for a further 24 h. Subsequently, the cells were inoculated with a loop from the Petri dish (culture 2) in 200 ml of a 2L shake flask with 2 baffles (composition analog plate and batch medium) and at 30 0 C on an orbital shaker at a speed of 250 rpm for Incubated for 24 h.
Der Inhalt der Schüttelkolben diente als Vorkultur zur Inokulation eines 75 L-Fermenter mit einem Füllvolumen von 50 L. Der pH-Wert wurde mit Hilfe von Ammoniakgas auf pH 6,8 geregelt. Die Begasungsrate betrug ca. 0,33 vvm.The contents of the shake flasks served as a preculture for the inoculation of a 75 L fermenter with a filling volume of 50 L. The pH was controlled with the aid of ammonia gas to pH 6.8. The fumigation rate was about 0.33 vvm.
Die Hauptkultur wurde in einem 5 m3-Kessel mit 700 L Füllvolumen in der Batch-Phase durchgeführt. Hierzu wurde nach weiteren 24 h die Kultur aus dem 75 L-Fermenter in den 5 m3-Kessel überführt. Der pH-Wert wurde mit Ammoniak auf pH 6,8 geregelt. Der gelöste Sauerstoff wurde im Bereich von 20 bis 30 % (Luftsättigung) durch Anpassung der Begasungsrate und Rührerdrehzahl geregelt.The main culture was carried out in a 5 m 3 kettle with a 700 L filling volume in the batch phase. After a further 24 h, the culture was transferred from the 75 L fermenter to the 5 m 3 kettle. The pH was adjusted to pH 6.8 with ammonia. The dissolved oxygen was controlled in the range of 20 to 30% (air saturation) by adjusting the gasification rate and stirrer speed.
Nach ca. 24 h sank die Glucosekonzentration des Batch-Mediums auf einen Wert unter 1 g/L und die Zudosierung des Feeds wurde gestartet. Nach ca. 80 h wurde das Endvolumen der Fermentation von ca. 3200 L erreicht. Die Endkonzentrationen in der Fermentation betrugen OD6io: 140, 1 ,5-Diaminopentan: 72 g/L, Lysin x HCl: 15 g/L, Acetyldiaminopentan: 10 g/L.After about 24 h, the glucose concentration of the batch medium dropped to a value below 1 g / L and the feed was started. After about 80 h, the final volume of the fermentation of about 3200 L was reached. The final concentrations in the fermentation were OD 6 io: 140, 1, 5-diaminopentane: 72 g / L, lysine x HCl: 15 g / L, acetyldiaminopentane: 10 g / L.
Die Zellabtrennung erfolgte unter Verwendung einer Vollmantelzentrifuge, wobei die Zellen im Trüblauf um den Faktor 3,3 aufkonzentriert wurden und die Trübung im Klar- lauf bei ca OD6io 5 lag. Der Klarlauf wurde mit 50%iger NaOH auf pH 13,5 eingestellt. Tabelle 2: Zusammensetzungen der MedienThe cell separation was carried out using a solid bowl centrifuge, the cells were concentrated in the Trüblauf by a factor of 3.3 and the turbidity was in the clear at ca OD 6 io 5. The clear liquor was adjusted to pH 13.5 with 50% NaOH. Table 2: Compositions of the media
Alle Medienbestandteile wurden bei 1210C für 30 min sterilisiert, mit Ausnahme der Vitamine, welche unter Verwendung eines 0,2 μm-Filters sterilfiltriert wurden.All media components were sterilized at 121 ° C. for 30 minutes, except for the vitamins, which were sterile filtered using a 0.2 μm filter.
Ausführungsbeispiel 1 : Gewinnung von DAP aus Fermentationsbrühe eines Lysin- und DAP-produzierenden MikroorganismusEmbodiment 1: Recovery of DAP from fermentation broth of a lysine and DAP producing microorganism
a) Durchführung der thermischen Behandlung und Extraktion (Einzel-Batch)a) Performance of the thermal treatment and extraction (single batch)
In einen 1 m3-Kessel aus Edelstahl wurden 750 kg (670 I) zellfreie, auf pH-Wert 13,5 mit NaOH eingestellte Fermentationsbrühe eingefüllt. Der Reaktorinhalt wurde auf Rückflusstemperatur (ca. 1030C) erhitzt und 5 Stunden lang refluxiert. Das dabei entstehende ammoniakhaltige Abgas wurde über einen Waschturm (mit Wasser als Waschflüssigkeit) abgefangen. Nach Abkühlen auf 60 0C wurden 140 kg n-Butanol zugefahren, 15 Minuten lang bei 600C nachgerührt und der Ansatz 2 Stunden absitzen gelassen. Nach der Phasentrennung wurde die wässrige Unterphase in einen 1 m3-Behälter abgelassen. Die organische Phase wurde in einem weiteren Behälter gesammelt. Die wässrige Phase wurde nach Zugabe von je 35 L VE-Wasser bei 600C weitere zwei Mal mit je 140 kg n-Butanol extrahiert.750 kg (670 l) of cell-free fermentation broth adjusted to pH 13.5 with NaOH were introduced into a stainless steel 1 m 3 kettle. The reactor contents were heated to reflux temperature (about 103 0 C) and refluxed for 5 hours. The resulting ammonia-containing waste gas was trapped via a scrubbing tower (with water as the scrubbing liquid). After cooling to 60 0 C 140 kg of n-butanol were added, stirred for 15 minutes at 60 0 C and the mixture allowed to settle for 2 hours. After phase separation, the aqueous lower phase was drained into a 1 m 3 tank. The organic phase was collected in another container. The aqueous phase was extracted after addition of 35 L of deionized water at 60 0 C for two more times with 140 kg of n-butanol.
Erhalten wurden insgesamt 590 kg organischer Extraktphasen, in denen 44,2 kg DAP enthalten waren (Gehalt ca. 7,5 %).A total of 590 kg of organic extract phases containing 44.2 kg of DAP were obtained (content about 7.5%).
b) Durchführung der Destillation:b) Carrying out the distillation:
In einem 1 m3-Edelstahl-Kessel mit aufgesetzter Kolonne (4 theoretische Trennstufen) wurden 900 kg der vereinten organischen Phasen aus der Extraktion (Teilschritt a) vorgelegt. Unter Abdestillation von Wasser/n-BuOH wurden bei konstantem Volumen weitere 1400 kg Extraktlösung zugefahren. Anschließend wurde der Druck auf 200 mbar abgesenkt und destilliert, bis 360 kg Sumpf verblieben.In a 1 m 3 stainless steel boiler with attached column (4 theoretical plates) 900 kg of the combined organic phases from the extraction (sub-step a) were submitted. While distilling off water / n-BuOH, a further 1400 kg of extract solution were added at constant volume. Subsequently, the pressure was lowered to 200 mbar and distilled until 360 kg sump remained.
In einem weiteren Edelstahl-Kessel mit 400 L Volumen und aufgesetzter Kolonne mit 8 theoretischen Trennstufen wurde dieser Sumpf bei 40 mbar weiter destilliert. Nach Abtrennung eines BuOH-Vorlaufs und einer Mischfraktion wurden insgesamt 155,4 kg DAP mit einer durchschnittlichen Reinheit von 99,6 Gew.-% überdestilliert Tetrahydro- pyridin als Nebenkomponente wurde in keiner Fraktion mit mehr als 0,4 % (GC-FI.) gefunden.In a further stainless steel vessel with 400 L volume and attached column with 8 theoretical plates, this bottom was further distilled at 40 mbar. After separation of a BuOH flow and a mixed fraction a total of 155.4 kg of DAP were distilled with an average purity of 99.6 wt .-% tetrahydropyridine as a minor component was in any fraction with more than 0.4% (GC-FI. ) found.
Testbeispiel 1 : Untersuchung der Geschwindigkeit des Stoffübergangs bei der ExtraktionTest Example 1: Investigation of the rate of mass transfer in the extraction
Die Einstellung des Verteilungsgleichgewichts eines erfindungsgemäß hergestellten organischen DAP-Extraktes wurde im 2,5-l-Reaktor unter intensivem Rühren bei 60 0C durch Entnahme einer zweiphasigen Probe zu verschiedenen Zeitpunkten über das Bodenventil und sofortige Trennung der Phasen nach Entmischung untersucht. Trotz einer gewissen Ungenauigkeit durch die erforderliche Absitzzeit der Probe kann ge- schlössen werden, dass die Verteilung sehr schnell erfolgt - die Probe nach 15 Sekunden wies 98,5 % des nach 5 Minuten gefundenen DAP auf. Eine Nachrührzeit von 15 Minuten ist somit als ausreichend anzusehen. Testbeispiel 2: Untersuchung der PhasentrennungsgeschwindigkeitThe adjustment of the distribution equilibrium of an organic DAP extract prepared according to the invention was investigated in the 2.5 l reactor with vigorous stirring at 60 ° C. by taking a two-phase sample at different times via the bottom valve and immediate separation of the phases after demixing. Despite some inaccuracy due to the required sample settling time, it can be concluded that the distribution is very fast - the sample after 15 seconds had 98.5% of the DAP found after 5 minutes. A stirring time of 15 minutes is therefore sufficient. Test Example 2: Study of the phase separation speed
Die Geschwindigkeit der Phasentrennung wurde in einem 2,5-l-Doppelmantelreaktor mit 3-stufigem Balkenrührer und 4 Stromstörern untersucht.The rate of phase separation was investigated in a 2.5 L jacketed reactor with 3-stage bar stirrer and 4 baffles.
Die Versuchsergebnisse für Ansätze (je 4 aufeinander folgende Extraktionen) mit und ohne thermische Behandlung sind in den Tabellen 3 und 4 zusammengefasst.The test results for batches (4 consecutive extractions) with and without thermal treatment are summarized in Tables 3 and 4.
Tabelle 3: Phasentrennzeiten bei Extraktion einer Brühe mit Hefeextrakt ohne thermi- sehe BehandlungTable 3: Phase separation times on extraction of a broth with yeast extract without thermal treatment
Emulsion als Zwischenphase Emulsion as intermediate phase
Tabelle 4: Phasentrennzeiten bei Extraktion einer Brühe mit Hefeextrakt nach thermischer Behandlung (4h, 104 0C)Table 4: Phase separation times on extraction of a broth with yeast extract after thermal treatment (4 h, 104 0 C)
Testbeispiel 3: Verkochung und Acetyl-DAP-Spaltung Test Example 3: Coking and acetyl-DAP cleavage
Es wurde beobachtet, dass der Produktionsorganismus einen Teil des gebildeten DAPs zusätzlich an einer der beiden Aminogruppen acetyliert.It has been observed that the production organism additionally acetylates a part of the DAP formed on one of the two amino groups.
Es konnte gezeigt werden, dass sich das Acetyl-Diaminopentan durch Rückflusskochen der auf einen pH-Wert oberhalb von 13 alkalisch gestellten Fermentationsbrühe unter Freisetzung des Diaminopentans verseifen lässt (s. Fig. 2). Dadurch kann die Ausbeute gesteigert werden. Die Verseifung unter sauren Bedingungen (pH 1 mit H2SO4) verläuft dagegen nur sehr langsam.It could be shown that the acetyl-diaminopentane can be saponified by refluxing the fermentation broth, which has been made alkaline to a pH above 13, with the release of the diaminopentane (see Fig. 2). This can increase the yield. Saponification under acidic conditions (pH 1 with H 2 SO 4 ) is very slow.
Beim Rückflusskochen wird bereits bei ca. 95 0C beginnender Rückfluss beobachtet, im weiteren Verlauf stellt sich eine Rückflusstemperatur von ca. 103-105 0C im Sumpf ein. Bei der Verseifung wird vor allem während des Hochheizens die Freisetzung von Ammoniak beobachtet (s. Fig. 3).When refluxing is already observed at about 95 0 C beginning reflux, in the further course, a reflux temperature of about 103-105 0 C sets in the sump. During saponification, the release of ammonia is observed, especially during the high-temperature heating (see Fig. 3).
Für die erfolgreiche Verkochung ist insbesondere ein pH-Wert von mindestens 13,5 von Vorteil.In particular, a pH of at least 13.5 is advantageous for successful boiling.
Testbeispiel 4: Abhängigkeit der Phasentrenngeschwindigkeit von der Dauer der thermischen BehandlungTest Example 4 Dependence of the phase separation rate on the duration of the thermal treatment
Jeweils 300 ml_ einer erfindungsgemäß hergestellten Fermentationsbrühe mit pH 13,0 wurden in einem 0,75-L Doppelmantelreaktor mit Impellerrührer nach der thermischen Behandlung 3 Mal bei 60 0C nach Rühren mit 350 U/min mit jeweils 100 ml_ n-BuOH (wassergesättigt) extrahiert. Die Ergebnisse sind in Tabelle 5 zusammengefasst. Tabelle 5: Absitzzeit nach verschiedener Dauer der thermischen BehandlungEach 300 ml of an inventively prepared fermentation broth with pH 13.0 3 times were at 60 0 C (water-saturated) after stirring at 350 revolutions / min with 100 ml of n-BuOH in a 0.75-L jacketed reactor with a impeller stirrer after thermal treatment extracted. The results are summarized in Table 5. Table 5: Absperrzeit after different duration of the thermal treatment
Testbeispiel 5: pH-Abhängigkeit des VerteilungskoeffizientenTest Example 5: pH dependence of the partition coefficient
Je 2 kg einer erfindungsgemäß hergestellten Fermentationsbrühe wurden mit DAP auf einen Gehalt von 10 % aufgestockt und der pH durch Zugabe von NaOH auf 1 1 ,0, 12,0 bzw. 13,5 eingestellt. Bei 60 0C wurde zur Bestimmung der Verteilungskoeffizienten jeweils fünf mal mit 150 g wassergesättigtem n-BuOH extrahiert. Die analytisch bestimmten DAP-Gehalte der wässrigen und organischen Phasen und die Verteilungskoeffizienten sind in Tabelle 6 angegeben.Each 2 kg of a fermentation broth produced according to the invention were increased to a content of 10% with DAP and the pH was adjusted to 1, 0, 12.0 or 13.5 by adding NaOH. At 60 0 C five times with water-saturated 150 g n-BuOH was extracted to determine the distribution coefficient. The analytically determined DAP contents of the aqueous and organic phases and the distribution coefficients are given in Table 6.
Tabelle 6: Verteilung des DAP zwischen Wasser und n-BuOH bei verschiedenen pH- WertenTable 6: Distribution of DAP between water and n-BuOH at different pH values
Testbeispiel 6: Abhängigkeit der Spaltungskinetik von AcDAP vom pH-Wert Test Example 6: Dependence of the Cleavage Kinetics of AcDAP on the pH
In einem 0,75L-Doppelmantelreaktor wurden je 500g erfindungsgemäß hergestellter Fermentationsbrühe mit hohem Gehalt an AcDAP durch Zugabe von 50%iger NaOH auf den gewünschten pH eingestellt und auf Rückflusstemperatur erhitzt. Nach 0,5, 1 , 2 und 4 h wurden Proben gezogen und per quantitativer HPLC vermessen. Die Ergebnisse sind in Tabelle 7 zusammengefasst.In a 0.75 L jacketed reactor, 500 g of fermentation broth with a high content of AcDAP prepared according to the invention were adjusted to the desired pH by addition of 50% strength NaOH and heated to reflux temperature. After 0.5, 1, 2 and 4 h samples were taken and measured by quantitative HPLC. The results are summarized in Table 7.
Tabelle 7: Abhängigkeit der Spaltungsgeschwindigkeit von AcDAP vom pH-WerlTable 7: Dependence of the cleavage rate of AcDAP on the pH value
VerkochAc- ung PH Lysin Ac-DAP DAP PH Lysin Ac-DAP DAP PH Lysin DAP DAPCulinary PH Lysine Ac-DAP DAP PH Lysine Ac-DAP DAP PH Lysine DAP DAP
[h] jeweils [Gew%] jeweils [Gew%] jeweils [Gew%][h] in each case [% by weight] in each case [% by weight] in each case [% by weight]
0 12,99 0 2,131 4,405 13,58 0 2,027 4,426 13,98 0 1 ,903 4,0510 12,99 0 2,131 4,405 13,58 0 2,027 4,426 13,98 0 1, 903 4,051
0,5 13,02 0 2,011 4,568 13,52 0 1 ,56 4,877 13,90 0 0,439 5,0130.5 13.02 0 2.011 4.568 13.52 0 1, 56 4.877 13.90 0 0.449 5.013
1 12,92 0 1 ,908 4,652 13,48 0 1 ,269 5,089 13,90 0 0,145 5,211 12,92 0 1, 908 4,652 13,48 0 1, 269 5,089 13,90 0 0,145 5,21
2 12,81 0 1 ,791 4,73 13,42 0 0,9 5,363 13,88 0 0 5,3272 12,81 0 1, 791 4,73 13,42 0 0,9 5,363 13,88 0 0 5,327
4 12,61 0 1 ,542 4,827 13,34 0 0,508 5,622 13,88 0 0 5,4344 12,61 0 1, 542 4,827 13,34 0 0,508 5,622 13,88 0 0 5,434
Testbeispiel 7: SalzfällungTest Example 7: Salt precipitation
Zu 130 g einer erfindungsgemäß hergestellten Extraktphase mit einem DAP-Gehalt von ca. 4.1 Gew.-% wurde bei 25-30 0C eine Lösung von 7,2 g Adipinsäure in 70 g wassergesättigtem Butanol zugetropft. Das Reaktionsgemisch wurde auf 3 0C abgekühlt, der Feststoff abfiltriert und über Nacht im Stickstoffstrom getrocknet. Erhalten wurden 12,7 g eines weißen Feststoffs, der per 13C-NMR als 1 :1 -Mischung von Adipinsäure und DAP charakterisiert wurde.To 130 g of an extract phase according to the invention with a DAP content of about 4.1 wt .-%, a solution of 7.2 g of adipic acid in 70 g of water-saturated butanol was added dropwise at 25-30 0 C. The reaction mixture was cooled to 3 0 C, the solid filtered off and dried overnight in a stream of nitrogen. 12.7 g of a white solid were obtained, which was characterized by 13 C-NMR as a 1: 1 mixture of adipic acid and DAP.
Auf die Offenbarung des hierin zitierten Standes der Technik wird in vollem Umfang Bezug genommen. The disclosure of the prior art cited herein will be fully incorporated by reference.

Claims

Patentansprüche claims
1. Verfahren zur Isolierung von 1 ,5-Diaminopentan (DAP) aus einer DAP-haltigen Fermentationsbrühe, wobei man die Fermentationsbrühe a) alkalisiert, b) ther- misch behandelt, c) DAP mit einem organischen Extraktionsmittel extrahiert, und d) DAP aus der abgetrennten organischen Phase isoliert.1. A process for the isolation of 1, 5-diaminopentane (DAP) from a DAP-containing fermentation broth, wherein the fermentation broth a) alkalized, b) treated thermally, c) DAP extracted with an organic extractant, and d) DAP isolated the separated organic phase.
2. Verfahren nach Anspruch 1 , wobei man die Fermentationsbrühe auf einen pH- Wert von >1 1 einstellt.2. The method of claim 1, wherein the fermentation broth is adjusted to a pH of> 1 1.
3. Verfahren nach Anspruch 2, wobei die pH-Wert-Einstellung durch Zugabe eines Alkali- oder Erdalkalimetallhydroxids erfolgt.3. The method of claim 2, wherein the pH adjustment is carried out by adding an alkali or alkaline earth metal hydroxide.
4. Verfahren nach einem der vorhergehenden Ansprüche, wobei man die alkalisier- te Fermentationsbrühe thermisch behandelt, indem man auf Rückflusstemperatur erhitzt.4. A process according to any one of the preceding claims wherein the alkalized fermentation broth is thermally treated by heating to reflux temperature.
5. Verfahren nach einem der vorhergehenden Ansprüche, wobei man die thermische Behandlung unter Bedingungen durchführt, die eine hydrolytische Spaltung von gegebenenfalls vorhandenem Acetyl-DAP bewirken.5. The method according to any one of the preceding claims, wherein the thermal treatment is carried out under conditions which cause a hydrolytic cleavage of optionally present acetyl-DAP.
6. Verfahren nach einem der vorhergehenden Ansprüche, wobei man die DAP- Extraktion mit einem dipolar-protischen organischen Lösungsmittel durchführt.6. The method according to any one of the preceding claims, wherein the DAP extraction is carried out with a dipolar protic organic solvent.
7. Verfahren nach Anspruch 6, wobei das Extraktionsmittel ein Alkanol oder Cyclo- alkanol ist7. The method of claim 6, wherein the extractant is an alkanol or cycloalkanol
8. Verfahren nach einem der Ansprüche 6 und 7, wobei man die Extraktion und/oder die anschließende Phasentrennung bei erhöhter Temperatur durch- führt.8. The method according to any one of claims 6 and 7, wherein one carries out the extraction and / or the subsequent phase separation at elevated temperature.
9. Verfahren nach einem der vorhergehenden Ansprüche, wobei man aus der Fermentationsbrühe vor der Alkalisierung zelluläre Bestandteile entfernt.9. The method according to any one of the preceding claims, wherein removed from the fermentation broth before alkalization cellular components.
10. Verfahren nach einem der vorhergehenden Ansprüche, wobei man die DAP- haltige Phase des Extraktionsschrittes destillativ aufreinigt oder DAP daraus ausfällt. 10. The method according to any one of the preceding claims, wherein the DAP-containing phase of the extraction step is purified by distillation or DAP precipitates therefrom.
1 1. Verfahren nach einem der vorhergehenden Ansprüche, wobei die Fermentationsbrühe aus der Fermentation eines Mikroorganismus in einem Kulturmedium stammt, das komplexe Medienbestandteile umfasst.1 1. The method according to any one of the preceding claims, wherein the fermentation broth comes from the fermentation of a microorganism in a culture medium comprising complex media components.
12. Verfahren zur fermentativen Herstellung von DAP, wobei man einen Lysin- produzierenden Mikroorganismus unter Lysin- und gegebenenfalls DAP- produzierenden Bedingungen kultiviert und das gebildete DAP unter Anwendung eines Verfahrens nach einem der Ansprüche 1 bis 11 isoliert.12. A process for the fermentative production of DAP, wherein cultivating a lysine-producing microorganism under lysine and optionally DAP-producing conditions and the formed DAP using a method according to one of claims 1 to 11 isolated.
13. Verfahren nach Anspruch 12, wobei die Fermentation in einem Kulturmedium durchgeführt wird, welches komplexe Medienbestandteile enthält.13. The method of claim 12, wherein the fermentation is carried out in a culture medium containing complex media components.
14. Verfahren nach einem der Ansprüche 12 und 13, wobei der Lysin-produzierende Mikroorganismus Lysin Decarboxylase-Aktivität enthält.14. The method according to any one of claims 12 and 13, wherein the lysine-producing microorganism contains lysine decarboxylase activity.
15. Verfahren nach Anspruch 14, wobei der Lysin-produzierende Mikroorganismus eine heterologe Lysin Decarboxylase enthält.15. The method of claim 14, wherein the lysine-producing microorganism contains a heterologous lysine decarboxylase.
16. Verfahren nach einem der Ansprüche 14 und 15, wobei der Lysin-produzierende Mikroorganismus heterologe Lysin Decarboxylase Gene enthält.16. The method according to any one of claims 14 and 15, wherein the lysine-producing microorganism contains heterologous lysine decarboxylase genes.
17. Verfahren nach einem der Ansprüche 12 und 13, wobei man die Lysin-haltige Fermentationsbrühe mit gegebenenfalls immobilisierter Lysin-Decarboxylase, in Kontakt bringt, um Lysin zu DAP zu decarboxylieren.17. The method according to any one of claims 12 and 13, wherein bringing the lysine-containing fermentation broth with optionally immobilized lysine decarboxylase, in contact to decarboxylate lysine to DAP.
18. Verfahren zur Herstellung eines DAP-haltigen Polymers, wobei man zunächst monomeres DAP nach einem Verfahren gemäß einem der Ansprüche 1 bis 17 fermentativ herstellt und isoliert und zusammen mit wenigsten einem weiteren Comonomer polymerisiert18. A process for the preparation of a DAP-containing polymer, wherein first preparing monomeric DAP by a process according to any one of claims 1 to 17 by fermentation and isolated and polymerized together with at least one other comonomer
19. Verfahren nach Anspruch 18, wobei das Comonomer ausgewählt ist unter Polyi- socyanaten und Polycarbonsäuren, sowie deren Salzen, Estern und Anhydriden.19. The method of claim 18, wherein the comonomer is selected from polyisocyanates and polycarboxylic acids, and their salts, esters and anhydrides.
20. Verfahren nach Anspruch 18 oder 19, wobei man wenigstens ein Comonomer dem isolierten DAP zusetzt oder ein Gemisch aus DAP und wenigstens einem Comonomer aus einer DAP-Fällung einsetzt. 20. The process according to claim 18 or 19, wherein at least one comonomer is added to the isolated DAP or a mixture of DAP and at least one comonomer from a DAP precipitate is used.
21. Verfahren nach Anspruch 20, wobei das DAP/Comonomer-Gemisch aus einer Salzfällung nach Anspruch 10 resultiert. 21. The method of claim 20, wherein the DAP / comonomer mixture results from a salt precipitation according to claim 10.
22. Verfahren nach Anspruch 21 , wobei das Comonomer eine Polycarbonsäure ist. 22. The method of claim 21, wherein the comonomer is a polycarboxylic acid.
EP09704118A 2008-01-23 2009-01-23 Method for fermentatively producing 1,5-diaminopentane Active EP2235194B1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PL09704118T PL2235194T3 (en) 2008-01-23 2009-01-23 Method for fermentatively producing 1,5-diaminopentane
EP09704118A EP2235194B1 (en) 2008-01-23 2009-01-23 Method for fermentatively producing 1,5-diaminopentane

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP08150575 2008-01-23
PCT/EP2009/050778 WO2009092793A2 (en) 2008-01-23 2009-01-23 Method for fermentatively producing 1,5-diaminopentane
EP09704118A EP2235194B1 (en) 2008-01-23 2009-01-23 Method for fermentatively producing 1,5-diaminopentane

Publications (2)

Publication Number Publication Date
EP2235194A2 true EP2235194A2 (en) 2010-10-06
EP2235194B1 EP2235194B1 (en) 2011-07-20

Family

ID=40793078

Family Applications (1)

Application Number Title Priority Date Filing Date
EP09704118A Active EP2235194B1 (en) 2008-01-23 2009-01-23 Method for fermentatively producing 1,5-diaminopentane

Country Status (8)

Country Link
US (1) US8906653B2 (en)
EP (1) EP2235194B1 (en)
JP (1) JP5646345B2 (en)
KR (1) KR101579994B1 (en)
CN (1) CN101981202B (en)
AT (1) ATE517190T1 (en)
PL (1) PL2235194T3 (en)
WO (1) WO2009092793A2 (en)

Families Citing this family (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2406710T3 (en) * 2008-01-31 2013-06-07 Basf Se Polyamide molding material [5,10] reinforced with fibers.
US8647642B2 (en) 2008-09-18 2014-02-11 Aviex Technologies, Llc Live bacterial vaccines resistant to carbon dioxide (CO2), acidic PH and/or osmolarity for viral infection prophylaxis or treatment
WO2011082976A1 (en) 2009-12-17 2011-07-14 Basf Se Method for purifying compounds comprising amino groups
JP5700575B2 (en) * 2010-03-01 2015-04-15 三井化学株式会社 Process for producing 1,5-pentamethylene diisocyanate
EP2684867B1 (en) * 2011-03-09 2019-01-02 Mitsui Chemicals, Inc. Pentamethylene diisocyanate, method for producing pentamethylene diisocyanate, polyisocyanate composition, polyurethane resin, and polyurea resin
KR101286158B1 (en) * 2011-08-24 2013-07-15 씨제이제일제당 (주) The seperation and purification method of 1,4-diaminobutane from a fermented solution
KR20140052189A (en) * 2012-10-22 2014-05-07 씨제이제일제당 (주) Refining method of organic amine, organic amine refined by the method, and polyamide prepared therefrom
EP2948558B1 (en) * 2013-01-28 2020-10-28 Cathay Biotech Inc. Purification of cadaverine using high boiling point solvent
US9914694B2 (en) 2013-01-28 2018-03-13 Cathay R&D Center Co., Ltd. Purification of cadaverine using high boiling point solvent
US9896409B2 (en) 2013-01-28 2018-02-20 Cathay R&D Center Co., Ltd. Purification of cadaverine using high boiling point solvent
EP3072880A4 (en) * 2013-11-19 2017-07-19 Toray Industries, Inc. 1, 5-pentamethylene diamine and method for producing same
KR20150115533A (en) * 2014-04-04 2015-10-14 씨제이제일제당 (주) Refining method of organic amine
US20170260554A1 (en) * 2014-07-31 2017-09-14 Karlsruher Institut für Technologie Method For The Enzyme-Catalyzed Production Of Prepolymers For Producing Plastics
AU2015369651B2 (en) 2014-12-23 2020-03-12 Genomatica, Inc. Method of producing and processing diamines
US10584149B2 (en) 2015-01-30 2020-03-10 Cathay Biotech Inc. Expression of Klebsiella oxytoca polypeptides involved in lysine decarboxylation, and methods and applications thereof
US20160297747A1 (en) 2015-04-08 2016-10-13 Invista North America S.A.R.L. Materials and methods for the selective recovery of monovalent products from aqueous solutions using continuous ion exchange
CN107709485B (en) 2015-04-20 2020-12-29 巴斯夫欧洲公司 Two-component coating mixture
KR101773135B1 (en) * 2015-05-08 2017-08-30 씨제이제일제당 주식회사 Refining method of 1,4-diaminobutane
CN107043330B (en) * 2016-02-06 2020-10-20 上海凯赛生物技术股份有限公司 Method for extracting 1, 5-pentamethylene diamine from solution system containing 1, 5-pentamethylene diamine salt
CN105861586B (en) * 2016-05-16 2017-05-24 宁夏伊品生物科技股份有限公司 Method for producing pentamethylene diamine by using carbon dioxide desorption process through fermentation
CN106367326B (en) * 2016-07-28 2018-10-30 南京工业大学 A kind of device of immobilized cell continuous production extraction pentanediamine
CN107779446B (en) * 2016-08-25 2021-09-07 上海凯赛生物技术股份有限公司 Immobilized lysine decarboxylase, preparation thereof, preparation method of 1, 5-pentanediamine and product
CN107779445B (en) * 2016-08-25 2021-02-19 上海凯赛生物技术股份有限公司 Immobilized lysine decarboxylase, preparation thereof, preparation method of 1, 5-pentanediamine and product
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria
US11129906B1 (en) 2016-12-07 2021-09-28 David Gordon Bermudes Chimeric protein toxins for expression by therapeutic bacteria
US10464880B2 (en) 2017-01-27 2019-11-05 Cathay Biotech Inc. Purification of cadaverine using high boiling point solvent
KR102094348B1 (en) * 2017-10-18 2020-03-27 씨제이제일제당 주식회사 Refining method of 1,5-diaminopentane
CN107880543A (en) * 2017-12-11 2018-04-06 苏州天亦辰针织有限公司 A kind of bio-based nylon facing material and its preparation technology
CN108997130A (en) * 2018-08-29 2018-12-14 南京工业大学 A kind of method 1,5- pentanediamine extraction and coupled at salt
EP3852923A1 (en) * 2018-09-18 2021-07-28 INVISTA Textiles (U.K.) Limited Systems and methods for recovering amines and their derivates from aqueous mixtures
CN109704978A (en) * 2018-12-25 2019-05-03 天津大学 A kind of purification process of pentanediamine
IT202100007331A1 (en) * 2021-03-25 2022-09-25 Univ Bologna Alma Mater Studiorum PROCEDURE FOR THE EXTRACTION OF POLYAMINE FROM NATURAL COMPLEX MATRICES
EP4353814A1 (en) 2021-05-19 2024-04-17 Asahi Kasei Kabushiki Kaisha Recombinant microorganism having diamine producing ability and method for manufacturing diamine
CN115611748A (en) * 2021-07-14 2023-01-17 上海凯赛生物技术股份有限公司 Separation method of 1, 5-pentanediamine

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5835197A (en) * 1981-08-26 1983-03-01 Kyowa Hakko Kogyo Co Ltd Plamid pcg 2
US4601893A (en) * 1984-02-08 1986-07-22 Pfizer Inc. Laminate device for controlled and prolonged release of substances to an ambient environment and method of use
GB2165546B (en) * 1984-08-21 1989-05-17 Asahi Chemical Ind A plasmid containing a gene for tetracycline resistance and dna fragments derived therefrom
JPH01131275A (en) * 1987-11-16 1989-05-24 Mitsui Petrochem Ind Ltd Polyamide composition
DE4027453A1 (en) 1990-08-30 1992-03-05 Degussa NEW PLASMIDES FROM CORYNEBACTERIUM GLUTAMICUM AND DERIVED PLASMIDE VECTORS
DE4440118C1 (en) * 1994-11-11 1995-11-09 Forschungszentrum Juelich Gmbh Gene expression in coryneform bacteria regulating DNA
JPH10229891A (en) 1997-02-20 1998-09-02 Mitsubishi Rayon Co Ltd Production of malonic acid derivative
CN1515538A (en) * 1997-02-20 2004-07-28 ������������ʽ���� Process for preparing malonate derivative
JP2002223771A (en) 2001-02-01 2002-08-13 Toray Ind Inc Method for producing cadaverine
JP4196620B2 (en) 2002-04-08 2008-12-17 東レ株式会社 Method for producing cadaverine for polyamide raw material
JP4356320B2 (en) 2003-01-08 2009-11-04 東レ株式会社 Method for producing cadaverine dicarboxylate and polyamide
JP2004222569A (en) * 2003-01-22 2004-08-12 Toray Ind Inc Corynebacterium, and cadaverine or salt thereof and method for producing the same
DE602004000428T2 (en) 2003-05-26 2006-10-19 Ajinomoto Co., Inc. Process for the preparation of cadaverine dicarboxylate and its use for the production of nylon
DE102004061846A1 (en) 2004-12-22 2006-07-13 Basf Ag Multiple promoters
WO2006123778A1 (en) * 2005-05-19 2006-11-23 Ajinomoto Co., Inc. Method for producing cadaverine carbonate, and method for producing polyamide using the same
CN102732578B (en) * 2006-03-30 2016-02-17 巴斯夫欧洲公司 Produce the method for cadaverine
WO2007135188A2 (en) 2006-05-24 2007-11-29 Evonik Degussa Gmbh Process for the preparation of l-methionine
JP2009545553A (en) * 2006-08-01 2009-12-24 ビーエーエスエフ ソシエタス・ヨーロピア Method for producing pentamethylene-1,5-diisocyanate
HUE028873T2 (en) * 2006-10-24 2017-01-30 Basf Se Method of increasing gene expression using modified codon usage
ES2539280T3 (en) * 2006-10-24 2015-06-29 Basf Se Procedure for reducing gene expression by using modified codons
RU2009128896A (en) 2006-12-29 2011-02-10 Эвоник Дегусса ГмБх (DE) METHIONINE SYNTHESIS WITH A REDUCED ABILITY FOR REACTION PRODUCT INHIBITION
DE102007005072A1 (en) * 2007-02-01 2008-08-07 Evonik Degussa Gmbh Process for the fermentative production of cadaverine
BRPI0807760A2 (en) 2007-02-19 2014-06-17 Evonik Degussa Gmbh CORINEFORM BACTERIA WITH FORMAT-THF SYNTETASE ACTIVITY AND / OR GLYCIN CLIVING ACTIVITY
WO2008101850A1 (en) 2007-02-19 2008-08-28 Evonik Degussa Gmbh Method of producing methionine in corynebacteria by over-expressing enzymes of the pentose phosphate pathway
JP2010530223A (en) 2007-06-15 2010-09-09 エボニック デグサ ゲーエムベーハー Microorganisms with deregulated vitamin B12 system
WO2009095440A1 (en) 2008-01-31 2009-08-06 Basf Se Transparent polyamide[5,10] molding compounds
ES2406710T3 (en) * 2008-01-31 2013-06-07 Basf Se Polyamide molding material [5,10] reinforced with fibers.
AU2009211870B2 (en) 2008-02-04 2013-09-05 Basf Se Method for the production of dipicolinate

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2009092793A2 *

Also Published As

Publication number Publication date
JP5646345B2 (en) 2014-12-24
WO2009092793A2 (en) 2009-07-30
US20100292429A1 (en) 2010-11-18
KR20100117084A (en) 2010-11-02
JP2011509679A (en) 2011-03-31
CN101981202B (en) 2013-09-11
CN101981202A (en) 2011-02-23
EP2235194B1 (en) 2011-07-20
WO2009092793A3 (en) 2009-10-15
PL2235194T3 (en) 2011-12-30
ATE517190T1 (en) 2011-08-15
KR101579994B1 (en) 2015-12-24
US8906653B2 (en) 2014-12-09

Similar Documents

Publication Publication Date Title
EP2235194B1 (en) Method for fermentatively producing 1,5-diaminopentane
EP1769080B1 (en) Microorganisms for producing sulphur-containing compounds
WO2008092720A1 (en) Process for production of cadaverine by fermentation
DE102005047596A1 (en) Process for the fermentative production of L-amino acids using coryneform bacteria
WO2004024932A2 (en) Method for zymotic production of fine chemicals (meta) containing sulphur
DE102004013503A1 (en) Process for producing L-amino acids using coryneform bacteria
EP1516059B1 (en) Method for the production of sulphur-containing fine chemicals by fermentation
EP1497443A2 (en) Methods for producing sulphurous fine chemicals by fermentation using meth-coding coryneform bacteria
DE10239308A1 (en) Fermentative production of sulfur-containing fine chemicals, useful e.g. as feed additive, by culturing bacteria containing heterologous sequence for methionine synthase
DE10109690A1 (en) New nucleotide sequences encoding the metY gene
CN100523208C (en) Method for zymotic production of fine chemicals (metY) containing sulphur
WO2007031479A1 (en) Method for producing amino acids using micro-organisms
DE102004009454A1 (en) Fermentative production of L-amino acids, especially methionine, useful e.g. in animal nutrition, by culturing bacteria in which components of the methionine uptake system are suppressed
DE10144493A1 (en) Process for the fermentative production of L-amino acids using coyneform bacteria
WO2005059143A1 (en) Pgro expression units
DE10114189B4 (en) Method for obtaining valuable substances from organisms by influencing / impairing the cell's own transport systems for these valuable substances or by using production strains which lack said transport systems
DE10021828A1 (en) New nucleotide sequences coding for the cdsA gene
DE10116519A1 (en) Preparation of pantothenic acid, useful especially as animal feed supplement, comprises fermenting coryneform bacteria that overexpress the glyA gene
DE10021831A1 (en) New nucleotide sequences coding for the fadD15 gene
DE10109688A1 (en) New nucleotide sequences coding for the metR and metZ genes
EP2940144A1 (en) Method for the production of l-lysine using an alkaliphilic bacterium
DE10021826A1 (en) New mutant coryneform bacterium, useful for production of amino acids, especially glutamate, has increased activity of cardiolipin synthase

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20100823

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA RS

GRAJ Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted

Free format text: ORIGINAL CODE: EPIDOSDIGR1

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RTI1 Title (correction)

Free format text: METHOD FOR FERMENTATIVELY PRODUCING 1,5-DIAMINOPENTANE

DAX Request for extension of the european patent (deleted)
GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

Free format text: NOT ENGLISH

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 502009000993

Country of ref document: DE

Effective date: 20110915

REG Reference to a national code

Ref country code: NL

Ref legal event code: VDEP

Effective date: 20110720

REG Reference to a national code

Ref country code: PL

Ref legal event code: T3

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111020

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111120

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111121

REG Reference to a national code

Ref country code: IE

Ref legal event code: FD4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111021

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: IE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

REG Reference to a national code

Ref country code: HU

Ref legal event code: AG4A

Ref document number: E012652

Country of ref document: HU

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

26N No opposition filed

Effective date: 20120423

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

BERE Be: lapsed

Owner name: BASF SE

Effective date: 20120131

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 502009000993

Country of ref document: DE

Effective date: 20120423

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120131

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20120928

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120131

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120131

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20111020

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20130123

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130131

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130131

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130123

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20110720

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120123

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 517190

Country of ref document: AT

Kind code of ref document: T

Effective date: 20140123

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20140123

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PL

Payment date: 20180105

Year of fee payment: 10

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190123

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: HU

Payment date: 20220104

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CZ

Payment date: 20220124

Year of fee payment: 14

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CZ

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230123

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230124

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20240129

Year of fee payment: 16